Fusion proteins for editing endogenous dna of eukaryotic cells

Abstract

A protein for editing endogenous DNA in a eukaryotic cell or eukaryotic organism at a target site comprising a site-specific endonuclease and a 5'-3' exonuclease, wherein the 5'-3' exonuclease is a monomeric 5'-3' exonuclease.

Description

Invention Field

[0001] This invention relates to proteins (such as fusion proteins) for editing endogenous DNA at target sites in eukaryotic cells or eukaryotic organisms, and nucleic acid molecules comprising polynucleotides encoding such proteins. The invention also relates to DNA constructs, plasmids, or vectors comprising the polynucleotides of said nucleic acid molecules. Furthermore, the invention relates to prokaryotic or eukaryotic cells comprising said proteins or nucleic acid molecules or DNA constructs, plasmids, or vectors. The invention further relates to kits for editing endogenous DNA at target sites in eukaryotic cells or eukaryotic organisms. Additionally, the invention relates to a method for inserting a target donor sequence of interest into the endogenous DNA of eukaryotic cells or eukaryotic organisms at a target site, and a method for modifying the endogenous DNA of eukaryotic cells or eukaryotic organisms at a target site. Cells or eukaryotic organisms produced by said method are also provided. Background of the Invention

[0003] Recent advances in genome editing using CRISPR / Cas technology have simplified targeted modifications of genes and the genome, opening new possibilities for genetic engineering. Site-specific endonucleases such as Cas9 make it relatively easy to induce targeted modifications in the genome because they can generate double-strand breaks (DSBs) at designated sites. DSBs are potentially detrimental to the genetic integrity of cells, and cells attempt to repair them through one of several endogenous repair pathways. In eukaryotes, the non-homologous end joining (NHEJ) pathway is the most common form of DSB repair. The NHEJ pathway can repair DSBs simply and rapidly at the expense of accuracy because the broken ends are directly joined together without the use of a DNA template for repair. Therefore, repair via NHEJ occasionally introduces mutations into the genome, typically deletions or insertions of several base pairs. Genetic engineering exploits this phenomenon to produce knockouts or knockdowns of target genes, as deletions or insertions of several base pairs can cause frameshift mutations or premature stop codons, which can produce mutant phenotypes.

[0004] However, genetic engineering requires more than just gene knockout or knockdown methods. It also requires methods to predictably knock nucleotide sequences into the genome or introduce specific mutations at target sites. Such modifications are not possible via the NHEJ pathway. However, DSB repair via the homology-mediated repair (HDR) pathway is well-suited for both sequence knock-in into the genome and gene substitution because HDR relies on a template for DNA break repair. Sequences located between the flanking arms of such a DNA repair template (also referred to herein as “donor nucleic acid,” or, if it is DNA, “donor DNA”) are integrated into the genome, provided that the flanking arms are homologous to the flanking sequences at the DSB site. Therefore, HDR allows for targeted integration and deletion of sequences within the genome. However, since NHEJ is the dominant repair pathway, DSB repair via the HDR pathway is rare in eukaryotic cells, and the threshold for its occurrence may be far below the practical limits for detection and isolation. This means that HDR-dependent gene editing applications only become feasible in eukaryotic cells if the frequency of DSB repair via the HDR pathway increases significantly. US20170175140 describes a method for increasing the frequency of homologous recombination in eukaryotic cells using 5'-exonucleases. However, the reported improvements are rather low, making it necessary to further improve the efficiency of HDR, and more broadly, the efficiency of gene editing, especially gene editing via the HDR mechanism.

[0005] Therefore, one object of the present invention is to provide methods and molecular tools thereof for improving the efficiency of HDR in gene editing in cells. Another object of the present invention is to provide methods and tools (e.g., proteins, nucleic acid molecules, and / or kits) for increasing the frequency of gene replacement events in eukaryotic cells for editing target nucleotide sequences at genomic target sites in a predictable manner. A further object of the present invention is to provide proteins or polynucleotides encoding them for gene editing in eukaryotic cells and / or organisms, particularly in plants. Invention Overview

[0007] These objectives are achieved according to the claims. In addition, these objectives are achieved by the following:

[0008] 1) A protein for editing endogenous DNA at a target site in eukaryotic cells or eukaryotic organisms, the protein comprising a site-specific endonuclease and a 5'-3' exonuclease, wherein the 5'-3' exonuclease is a monomeric 5'-3' exonuclease.

[0009] 2) A protein for editing endogenous DNA at a target site in eukaryotic cells or eukaryotic organisms, said protein comprising a site-specific endonuclease and a 5'-3' exonuclease, wherein said 5'-3' exonuclease is a monomeric 5'-3' exonuclease having a concentration of at least 0.072 (μM·s) in the in vitro exonuclease assay described in the specification. -1 The catalytic efficiency k of 5'-3' exonuclease cat / K m Or at least 0.50s -1 The turnover number.

[0010] 3) The protein according to item 1 or 2, wherein the 5'-3' exonuclease is:

[0011] In the in vitro exonuclease assay described in the instructions, at the catalytic efficiency k cat / K m Or, in terms of the number of transformations, it has the same or higher 5'-3' exonuclease activity compared to T7 exonuclease (SEQ ID NO: 30); or

[0012] In the in vitro exonuclease assay described in the instructions, at the catalytic efficiency k cat / K m In terms of the number of transitions, it has at least twice the 5'-3' exonuclease activity of T5 exonuclease (SEQ ID NO: 31).

[0013] 4) The protein according to any one of items 1, 2 and 3, wherein the protein is a fusion protein comprising the site-specific endonuclease and the 5'-3' exonuclease.

[0014] 5) The protein according to any one of items 1, 2 and 3, wherein the protein is an oligomeric protein (protein complex) comprising a first protein subunit and a second protein subunit, the first protein subunit comprising the exonuclease, and the second protein subunit comprising the exonuclease.

[0015] 6) The protein according to claim 5, wherein the first subunit comprises (as a domain of the first subunit) the site-specific endonuclease and a first interacting domain, the second subunit comprises (as a domain of the second subunit) the 5'-3' exonuclease and a second interacting domain, wherein the first interacting domain and the second interacting domain bind to each other to form the oligomeric protein (protein complex).

[0016] 7) The protein according to any one of items 1 to 6, wherein the 5'-3' exonuclease is a protein with the following amino acid sequence or containing the following amino acid sequence:

[0017] (i) The amino acid sequence of SEQ ID NO: 30 (T7 exonuclease); or

[0018] (ii) An amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 30; or

[0019] (iii) An amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 30; or

[0020] (iv) An amino acid sequence having 1 to 50 amino acid substitutions, additions, deletions and / or insertions compared to the amino acid sequence of SEQ ID NO: 30.

[0021] 8) The fusion protein according to any one of items 1 to 6, wherein the 5'-3' exonuclease is a protein with the following amino acid sequence or containing the following amino acid sequence:

[0022] (i) The amino acid sequence of SEQ ID NO: 32 (UL12-1 exonuclease) or SEQ ID NO: 33 (UL12-2 exonuclease); or

[0023] (ii) An amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33; or

[0024] (iii) An amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33; or

[0025] (iv) An amino acid sequence having 1 to 120 amino acid substitutions, additions, deletions and / or insertions compared to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33.

[0026] 9) The protein according to any one of claims 1 to 8, wherein the site-specific endonuclease is a CRISPR nuclease capable of inducing DNA double-strand breaks, such as Cas9, or a nicking enzyme variant of a CRISPR nuclease, such as Cas9, capable of inducing single-strand nicks in double-stranded DNA.

[0027] 10) The protein according to any one of claims 1 to 9, wherein the protein provides a higher frequency of gene-targeting events compared to the individual application of the site-specific endonuclease and the 5'-3' exonuclease of the protein when they are provided together with the donor nucleic acid without fusing together or forming a protein complex.

[0028] 11) The fusion protein according to item 4, optionally as further defined in any one of items 2, 3 and 7 to 10, wherein the 5'-3' exonuclease is fused to the N-terminus or C-terminus of the site-specific endonuclease.

[0029] 12) The fusion protein according to item 4, optionally as further defined in any one of items 2, 3 and 7 to 11, wherein the site-specific endonuclease and the 5'-3' exonuclease are fused via a polypeptide linker.

[0030] 13) The fusion protein according to item 12, wherein the polypeptide linker consists of 5 to 300 amino acid residues, preferably 10 to 200 amino acid residues, and more preferably 20 to 120 amino acid residues.

[0031] 14) A nucleic acid molecule comprising a protein encoding any one of items 1 to 13, preferably a polynucleotide encoding a protein encoding any one of items 4 and 11 to 13.

[0032] 15) Nucleic acid constructs, plasmids or vectors comprising polynucleotides of the nucleic acid molecules described in item 14.

[0033] 16) A kit containing:

[0034] -A nucleic acid molecule containing a polynucleotide encoding the first protein subunit as described in item 5 or 6, and

[0035] - A nucleic acid molecule containing a polynucleotide encoding the second protein subunit as described in item 5 or 6.

[0036] 17) Prokaryotic or eukaryotic cells, which contain:

[0037] i) The protein described in any one of items 1 to 13,

[0038] ii) The nucleic acid molecules described in item 14,

[0039] iii) The nucleic acid constructs, plasmids, or vectors described in item 15, or

[0040] The kit described in item 16 of section iv).

[0041] 18) The cell according to item 17, wherein the cell is a eukaryotic cell and further comprises donor nucleic acid for homology-mediated DNA repair.

[0042] 19) The cell according to item 18, wherein the donor nucleic acid comprises, in the following order:

[0043] - A first homologous arm, which is homologous to a first region flanking the target site on the first side of the target site in the cell genome.

[0044] - An optional target donor sequence, which is inserted into the genomic DNA of the cell at the target site, and

[0045] - A second homologous arm, which is homologous to a second region on the second side of the target site.

[0046] 20) The cell according to item 19, wherein the donor sequence comprises, preferably, up to 10,000 nucleotides in length, preferably composed thereof.

[0047] 21) A cell according to any one of claims 17 to 20, further comprising guide RNA or a guide construct encoding said guide RNA, said guide RNA being capable of binding site-specific endonucleases and guiding the site-specific endonucleases to target sites in the genome of said cell.

[0048] 22) The cell according to any one of items 17 to 21, wherein the cell is a plant cell.

[0049] 23) Non-human objects, preferably plants, which include cells as described in any one of items 17 to 22.

[0050] 24) A kit for editing endogenous DNA at a target site in eukaryotic cells or eukaryotic organisms, said kit comprising:

[0051] (a) the donor nucleic acid as defined in item 19 or a donor construct containing said donor nucleic acid, and

[0052] (b) The protein as defined in any of items 1 to 13, or the nucleic acid as defined in item 14, or the DNA construct, plasmid or vector described in item 15, or the kit described in item 16.

[0053] 25) The kit according to item 24 further comprises:

[0054] (c) Cells or eukaryotic organisms.

[0055] 26) The kit according to item 24 or 25 further comprises:

[0056] (d) A guide RNA (gRNA) that can bind to the site-specific endonuclease and guide the protein to a target site on the endogenous DNA of the cell or organism; or a nucleic acid molecule encoding the guide RNA.

[0057] 27) A method for modifying endogenous DNA in a eukaryotic cell or eukaryotic organism at a target site, the method comprising providing the cell or organism with:

[0058] (a) The donor nucleic acid as defined in item 19, and

[0059] (b) The protein as defined in any of items 1 to 13, or the nucleic acid as defined in item 14, or the DNA construct, plasmid, or vector described in item 15, or the kit described in item 16.

[0060] The modification of the endogenous DNA includes inserting a target donor sequence into the endogenous DNA at the target site via homology-mediated repair, and / or includes deleting a sequence in the endogenous DNA at the target site via homology-mediated repair.

[0061] 28) The method according to item 27, wherein the method is a method of inserting a target donor sequence into the endogenous DNA of a eukaryotic cell or eukaryotic organism at a target site, wherein the target donor sequence contained in the donor nucleic acid is inserted into the endogenous DNA.

[0062] 29) The method according to any one of claims 28 further comprises providing the cell or organism with:

[0063] (c) A guide RNA (gRNA) or a nucleic acid encoding the guide RNA (guide nucleic acid), wherein the gRNA is capable of binding to the site-specific endonuclease and of guiding the protein to the target site in the endogenous DNA of the cell or organism.

[0064] 30) The method according to item 28 or 29, wherein the donor sequence has a length of up to 15 kbp.

[0065] 31) Use of the fusion protein according to any one of items 1 to 13 for gene editing in eukaryotic cells.

[0066] 32) Cells or eukaryotic organisms produced by any one of items 27 to 30.

[0067] Preferred embodiments that can be combined with the technical solutions defined in the claims or the preceding items are described in the following detailed description.

[0068] This invention is based on the surprising discovery that 5'-3' exonucleases (i.e., exonucleases that hydrolyze DNA in the 5' to 3' direction) vary greatly in their ability to increase HDR frequency and therefore in the efficiency of gene editing via HDR. Notably, the inventors have found that monomeric 5'-3' exonucleases exhibiting high in vitro 5'-3' exonuclease activity are particularly well-suited for significantly increasing HDR. In contrast, monomeric enzymes with too low 5'-exonuclease activity or polymeric enzymes are not suitable for increasing HDR frequency. The inventors have further discovered (even among monomeric 5'-3' exonucleases) that 5'-3' exonucleases vary greatly in their ability to increase HDR frequency and therefore in the efficiency of gene editing via HDR. Furthermore, the inventors have discovered that by combining a nuclease with a specific 5'-3' exonuclease exhibiting high activity against single-stranded or double-stranded breaks (e.g., blunt ends or staggered ends) produced by said nuclease, the efficiency of gene editing via HDR can be improved. Therefore, the present invention provides improved methods, proteins, kits, and nucleic acid molecules for gene editing via HDR. Consequently, the present invention makes HDR competitive with other faster NHEJ pathways for DSB repair in cells, particularly eukaryotic cells.

[0069] Brief description of the attached figures

[0070] Figure 1 Identification of putative single guide RNA (sgRNA) target sites in the PAM-in direction near the stop codon in the NbPGK (Nicotiana benthamiana phosphoglycerate kinase) genomic sequence (using CRISPOR and CRISPR-P v2.0), see Example 1. LB represents the left boundary of T-DNA; RB represents the right boundary of T-DNA; GUS is the ORF encoding the GUS protein; 5'HA and 3'HA represent the binding regions of the homologous arms on the donor DNA and the 5' and 3' homologous arms of the donor DNA on the genomic DNA. Arrows indicate the location of the sgRNA target.

[0071] Figure 2Design of SpCas9 exonuclease fusion and sgRNA. A: N-terminal Exo-Cas9 fusion. B: C-terminal Cas9-Exo fusion. Details are explained in Example 2. Exo represents the exonuclease-coding fragment; the adapter represents the adapter between the Exo and SpCas9i fragments; tOCS is the transcription terminator. N-SpCas9i-N represents a Cas9 version with two NLS signals (nuclear localization signals). The vertical box labeled "BsaI" represents the IIS type restriction endonuclease recognition site of the restriction enzyme BsaI; the horizontal box containing the base quadruplet represents the BsaI cleavage site that forms the GG overhang, used to assemble adjacent fragments by ligation using the Golden Gate (GG) cloning method described by Marillonnet et al. (especially in WO2011154147A1).

[0072] Figure 3 : Schematic diagram of sgRNA construct design using the Golden Gate (GG) cloning method. SlU6 represents the U6 promoter (SlU6, pAGT5824) from tomato (Solanum lycopersicum).

[0073] Figure 4: Results of gene targeting transiently expressed in *Tobacco Benedict* using a translational GUS fusion with NbPGK. A) Relative GUS activity 4 days after inoculation with the gene-targeting construct. Different exonuclease fusions with Cas9 produced different GUS activities. GUS activity is relative to the Act2p-GUS control construct. Relative GUS activity is expressed per μg of total protein. E-4LF2-Cas9 represents the N-terminal fusion, where exonuclease E is on the N-terminal side of Cas9 and linked via adapter 4LF2. Cas9-4LF2-E represents the C-terminal fusion, where exonuclease E is on the C-terminal side of Cas9 and linked via adapter 4LF2. Each column is labeled with the abbreviation of the exonuclease of the fusion protein. UL12 refers to UL12-1 exonuclease. DU represents DUMAS exonuclease. MD represents MD5 exonuclease. dCas9 represents inactivated Cas9. B) This is a replication of the experiment shown in A), except for the fusion of Cas9 with λ-exonuclease (LaExo) (a multimeric (trimeric) exonuclease), indicating that the fusion does not lead to an increase in HDR. C) Individual expression of Cas9 and UL12 was tested using the same assays as in A) and B), showing that they lead to an increase in HDR, but not as strongly as the fusion. D) Stained leaf discs from quantitative GUS measurements in A). Magnified leaf discs compare leaf discs with high GUS activity inoculated with the Cas9::UL12 fusion (left) to a Cas9 control without exonuclease (right).

[0074] Figure 5 : Analysis of exonuclease activity by in vitro processing of blunt-terminated hairpin oligonucleotides. T5 and T7 exonuclease activities were compared. A) Based on Nikiforov (2014), but with 5' phosphorylation and carrying Oregon green fluorescent dye (SEQ ID NO: 37). Stacking of the dye with the final G:C base pair quenched the fluorescence. After exonuclease degradation from the 5' end, stacking did not occur and quenching was lifted, resulting in higher fluorescence. B) Fluorescence was measured over 30 minutes at 27°C from two of the four concentrations of hairpin oligonucleotides (10 and 20 μM). C) Fluorescence was measured for 5 μM hairpin oligonucleotides and three enzymes (T5: T5-exonuclease; T7: T7-exonuclease; LaExo: λ-exonuclease; H2O: water control) as described in B).

[0075] Figure 6A transgenic *Nicotiana benthamiana* tobacco mosaic virus (TMV) reporter line using GFP encoded in gDNA. This reporter line was used to determine the frequency and efficiency of HDR in Examples 7 through 22. "gDNA(nbi775)" represents the insert cassette in the transgenic *Nicotiana benthamiana* line as given in SEQ ID NO: 99, encoding a TMV with a truncated RdRP containing GFP, under the control of Act2p as a transcription promoter. GFP replaces the capsid protein (CP) of the TMV. MP stands for TMV movement protein. "Donor" represents the donor nucleic acid containing the donor sequence encoding the RNA-dependent RNA polymerase (RdRP) of the TMV, and is given in SEQ ID NO: 74. The RdRP was repaired by the HDR insert donor, allowing transcription of the replicative TMV expressing GFP. See Example 7 for details. Figure 6 The sequence segment of the TMV transgenic construct at the bottom is given as SEQ ID NO: 73.

[0076] Figure 7 Cas9 fused with an exonuclease resulted in increased HDR in plants. Cas9-4LF2-X indicates the use of a 4LF2 linker to fuse an exonuclease to the C-terminus of Cas9. Cas9WT and inactivated Cas9 (dCas9) were used as controls. Donor DNA such as... Figure 6 As shown. Exo1: Arabidopsis exonuclease I; LaExo: λ phage exonuclease; T5: phage T5 exonuclease; T7: phage T7 exonuclease; Exo3: exonuclease III from Escherichia coli; TREX1: triple-primer repair exonuclease 1 from Homo sapiens. See Example 8 for details.

[0077] Figure 8 HDR events were quantified using GFP spot count. See Example 9 for details.

[0078] Figure 9 Genotyping of HDR events was performed by PCR (see Example 10). "Donor" represents donor nucleic acid. Primer pair P1 consists of primers 1F and 1R, primer pair P2 consists of primers 2F and 2R, and primer pair P3 consists of primers 3F and 3R. "NbWT" represents wild-type Nicotiana benthamiana DNA. "Cas9" represents wild-type Cas9, and "dCas9" represents inactivated Cas9.

[0079] Figure 10The exonuclease domain of Exo1 (Exo1ΔC) fused with Cas9 only slightly increases HDR efficiency (see Example 11). X-4LF2-Cas9 indicates the use of a 4LF2 linker to fuse the exonuclease to the N-terminus of Cas9. “Cas9” represents wild-type Cas9, and “dCas9” represents inactivated Cas9.

[0080] Figure 11 Comparison of UL12 homologs in HDR (see Example 12). X-4LF2-Cas9 indicates the use of a 4LF2 linker to fuse the exonuclease to the N-terminus of Cas9. “Cas9” represents wild-type Cas9, and “dCas9” represents inactivated Cas9. The amino acid sequence of UL12 is given in SEQ ID NO: 32. The amino acid sequences of other exonucleases are given below: UL12-2 = SEQ ID NO: 33, BGLF5 = SEQ ID NO: 34, Dumas = SEQ ID NO: 35, MD5 = SEQ ID NO: 36, PapE = SEQ ID NO: 43, PiE = SEQ ID NO: 44, SOX = SEQ ID NO: 68, AB4P = SEQ ID NO: 69.

[0081] Figure 12 In GFP spot counting analysis, the UL12 homologs PapE and PiE, fused with Cas9, showed increased HDR efficiency compared to UL12 (see Example 12). The amino acid sequences are given in the following SEQ ID NOs: UL12 = SEQ ID NO:32, UL12-2 = SEQ ID NO:33, PapE = SEQ ID NO:43, PiE = SEQ ID NO:44, AB4P = SEQ ID NO:69, MD5 = SEQ ID NO:36, Dumas = SEQ ID NO:35, BGLF5 = SEQ ID NO:34, SOX = SEQ ID NO:68.

[0082] Figure 13 Comparison of T7-homophores in HDR (see Example 13). The amino acid sequences are given in the following SEQ ID NO: UL12 / UL12-1 = SEQ ID NO:32, UL12-2 = SEQ ID NO:33, T7 = SEQ ID NO:30, ME15 / IME15 = SEQ ID NO:45, O3-12 / YerO3-12 = SEQ ID NO:70, SpiPh = SEQ ID NO:46, PHBO2 = SEQ ID NO:71, RaTL1 / RaITL1 = SEQ ID NO:72.

[0083] Figure 14 The T7 homolog ME15 exhibits HDR efficiency comparable to UL12 (see Example 13). The amino acid sequences are given in the following SEQ ID NOs: UL12 / UL12-1 = SEQ ID NO:32, UL12-2 = SEQ ID NO:33, T7 = SEQ ID NO:30, ME15 / IME15 = SEQ ID NO:45, O3-12 / YerO3-12 = SEQ ID NO:70, SpiPh = SEQ ID NO:46, PHBO2 = SEQ ID NO:71, RaTL1 / RaITL1 = SEQ ID NO:72.

[0084] Figure 15 Tree diagram of UL12 homologs (see Example 12).

[0085] Figure 16 Tree diagram of T7 homologs (see Example 13).

[0086] Figure 17 Amino acid sequence identity of UL12- and T7- homologs along with T5 (see Examples 12 and 13).

[0087] Figure 18 Comparison of exonuclease activity of monomeric exonucleases using blunt-ended DNA substrates (see Example 14). Hairpin oligomer: SEQ ID NO: 37.

[0088] Figure 19 Using blunt-ended DNA substrate SEQ ID NO: 37, the exonuclease activities of monomeric exonucleases (T5 and T7) and trimeric exonuclease (LaExo) were compared (see Example 15).

[0089] Figure 20 Co-expression of nuclear-localized LaExo (referred to as N-LaExo or LaExo-N) does not increase the activity of LaExo fused with Cas9 (see Example 16). Cas9-2LF2-X indicates the use of a 2LF2 linker to fuse the exonuclease to the C-terminus of Cas9. N-LaExo indicates the fusion of the nuclear localization signal (NLS) to the N-terminus of LaExo (λ exonuclease). LaExo-N indicates the fusion of the nuclear localization signal (NLS) to the C-terminus of LaExo. “Cas9” represents wild-type Cas9, and “dCas9” represents inactivated Cas9.

[0090] Figure 21 Determination of the minimum length of the homologous arm (see Example 17).

[0091] Figure 22 Cas12a-exonuclease fusion resulted in increased HDR in plants (see Example 18). X-4LF2-Cas12a indicates the use of a 4LF2 linker to fuse the exonuclease to the N-terminus of Cas12a. “Cas12a” represents wild-type Cas12a.

[0092] Figure 23 The HDR efficiency of the Cas12a exonuclease fusion protein was estimated by GFP dot counting (see Example 19). Labels were used as follows. Figure 22 As shown. dCas12a refers to inactivated Cas12a.

[0093] Figure 24 Comparative analysis of the cleavage patterns of Cas9- and Cas12a- exonuclease fusion proteins (see Example 20). X-Cas9 and X-Cas12a represent the fusion of the tested exonucleases into the N-terminus of Cas9 and Cas12a, respectively.

[0094] Figure 25A -D: Amplicon sequencing using Cas9 exonuclease fusion protein and PAM-out sgRNA (see Example 21).

[0095] Figure 26A -D: Amplicon sequencing using Cas12a-exonuclease fusion protein and crRNA in the PAM-out direction (see Example 22).

[0096] Figure 27A / B / C: Sequence alignment of the tested basic exonucleases homologous to UL12 (see Example 23). Residues with 5'-phosphate coordination and residues of the catalytic triad are indicated in bold and underline, respectively. Conserved motifs of basic exonucleases are underlined according to Goldstein and Weller (1998) and Buisson et al., 2009. UL12-group-specific motifs and PapE-specific amino acid residues are indicated by rectangles and lines, respectively. The amino acid sequence portions shown are as follows: BGLF5 = SEQ ID NO:34, SOX = SEQ ID NO:68, MD5 = SEQ ID NO:36, DUMAS = SEQ ID NO:35, AB4P = SEQ ID NO:69, PiE = SEQ ID NO:44, PapE = SEQ ID NO:43, UL12-1 = SEQ ID NO:32, UL12-2 = SEQ ID NO:33. The lines marked "shared" are not amino acid sequences, but indicate highly conserved positions in the sequence above.

[0097] Figure 28A / B: Amino acid sequence alignment of the sequence portion homologous to PapE (see Example 24). PapE-group specific motifs I, II, III, and IV are associated with increased HDR efficiency. The PapE-group is a subgroup of UL12-group exonucleases. PapE-group specific motifs I, II, III, and IV are shown. General basic exonuclease functional motifs II, III, and IV (according to Goldstein and Weller 1998 and Buisson et al., 2009) are indicated by the central bar. The amino acid sequence of UL12 is given in SEQ ID NO: 32, and the amino acid sequence of PapE is given in SEQ ID NO: 43. The amino acid sequences of proteins / peptides other than UL12 and PapE are given in SEQ ID NO: 75 to 94. Lines marked "common" are not amino acid sequences but indicate highly conserved positions in the sequences above.

[0098] Figure 29 : Alignment of the tested T7 exonuclease homologs. Residues of the catalytic triplet are indicated by bold underlines. T7 exonuclease-specific motifs I and II are indicated by lines. ME15-specific motifs I and II are also indicated by bars in the fifth section from the top. RalTL1 = SEQ ID NO:72, PaPHBO2 = SEQ ID NO:71, SpiPhage = SEQ ID NO:46, YerO3-12 = SEQ ID NO:70, T7 = SEQ ID NO:30, ME15 = SEQ ID NO:45. The lines marked "common" are not amino acid sequences, but indicate highly conserved positions in the sequences above. Invention Details

[0100] The protein and fusion protein of the present invention

[0101] The protein of this invention comprises a site-specific endonuclease (also referred to herein as "endonuclease") and a 5'-3' exonuclease (also referred to herein as "exonuclease"). An endonuclease is a protein possessing endonuclease activity and capable of cleaving phosphodiester bonds within a polynucleotide chain at a specific site. An exonuclease is a protein possessing 5'-3' exonuclease activity and capable of cleaving nucleotides from the ends of a polynucleotide chain in a 5' to 3' orientation. Therefore, both endonucleases and exonucleases are enzymes, and these enzymatic activities must be present in the protein of this invention. Therefore, the protein of this invention is a 5'-3' exonuclease and a site-specific endonuclease.

[0102] There are various possibilities regarding how endonucleases and exonucleases can be combined to form the protein of the present invention. They can be covalently or non-covalently bound. One example of covalent binding is a fusion protein comprising both an endonuclease and an exonuclease as fusion protein domains. Alternatively, the endonuclease and exonuclease can bind via other covalent chemical bonds such as disulfide bridges or via chemical linkers (e.g., using glutaraldehyde, optionally followed by reduction, such as using sodium borohydride). In the covalent binding between endonucleases and exonucleases, fusion proteins are preferred.

[0103] Alternatively, the protein may be an oligomeric protein (protein complex) comprising a first subunit (preferably a protein subunit) and a second subunit (preferably a protein subunit), wherein the first subunit comprises the endonuclease and the second subunit comprises the exonuclease. The first subunit may comprise:

[0104] - Site-specific endonucleases (e.g., domains as the first subunit) and

[0105] - First interacting domain (e.g., first protein interacting domain) or first interacting nucleic acid (e.g., nucleic acid containing aptamer);

[0106] The second subunit may include:

[0107] -5'-3' exonucleases (e.g., domains as the second subunit) and

[0108] - Second interaction domain (second protein or peptide interaction domain);

[0109] The first interacting domain or the first interacting nucleic acid and the second interacting domain bind to each other to form the oligomeric protein (protein complex). Therefore, in this embodiment, 5'-3' exonucleases and endonucleases can be brought into close proximity through specific protein-protein or protein-RNA interaction domains. For example, specific protein-protein interactions between peptide epitopes and single-chain antibodies that recognize the peptide have been used to generate strong transcriptional activators based on dCas9 (inactivated Cas9) (Tanenbaum et al., 2014, Cell, 159:635–646). Therefore, the first interacting domain can be such a peptide epitope, and the second interacting domain can be a single-chain antibody that binds to the peptide epitope. Similarly, coil-coil protein-protein interaction domains can be used for the same purpose (Lebar et al., Nat Chem Biol, 16:513–519). Therefore, the first and second interacting domains can be coil-coil protein-protein interaction domains.

[0110] Similarly, protein-RNA interaction domains can be used for the same purpose. Therefore, by inserting an aptamer into the gRNA used in this invention, a peptide that specifically recognizes the aptamer will bind to the gRNA and become physically close to the endonuclease (Ma et al., Nature Biotech, 34:528-531). This principle can be used to bring 5'-3' exonucleases into close proximity to endonucleases, for example, by fusing an exonuclease to a peptide that recognizes a specific RNA aptamer and fusing an RNA aptamer to gRNA. In a later embodiment, the protein of this invention is a CRISPR-Cas nuclease, the gRNA contains the aptamer, and the exonuclease contains a peptide that binds the aptamer as an interaction domain. Therefore, the aptamer-peptide complex can act as a non-covalent linker between the endonuclease and the exonuclease. In this embodiment, the protein may be an oligomeric protein comprising the following:

[0111] - The first subunit, which is a nuclease or contains a nuclease.

[0112] - The second subunit contains the 5'-3' exonuclease and (as the second interacting domain) a peptide capable of binding the aptamer, and

[0113] - A nucleic acid having a segment capable of binding an endonuclease (hereinafter referred to as "interacting nucleic acid," such as gRNA) and an aptamer (segment) capable of binding a 5'-3' exonuclease (particularly the peptide capable of binding the aptamer). As mentioned above, the endonuclease is preferably a CRISPR-Cas nuclease.

[0114] Therefore, the specific implementation scheme for the formation of a protein complex by endonuclease and exonuclease is as follows: the first interaction domain is a single-chain antibody, and the second interaction domain is a peptide epitope that specifically binds to the single-chain antibody; the second interaction domain is a single-chain antibody, and the first interaction domain is a peptide epitope that specifically binds to the single-chain antibody; the first interacting nucleic acid is a gRNA containing an aptamer, and the second interaction domain is a peptide that specifically recognizes and binds to the aptamer.

[0115] In the covalent and non-covalent binding between endonucleases and exonucleases, covalent binding is preferred, and fusion proteins containing endonucleases and exonucleases are more preferred.

[0116] The fusion protein of this invention is a fusion of a site-specific endonuclease and a 5'-3' exonuclease. Endonuclease and exonuclease represent the domains of the fusion protein. Hereinafter, when referring to endonuclease or exonuclease in the context of a fusion protein, it refers to the endonuclease domain or exonuclease domain of the fusion protein, respectively. The exonuclease may be fused to the N-terminus or C-terminus of the site-specific endonuclease. Fusion can be direct, i.e., without a linker. However, preferably, the two domains are fused via a linker polypeptide to avoid steric hindrance between and / or between the two domains. The fusion protein is a 5'-3' exonuclease and a site-specific endonuclease (and these functions are typically present in individual domains of the fusion protein).

[0117] The linker is a polypeptide with at least 10, preferably at least 20, and more preferably at least 30 amino acid residues. The maximum number of amino acid residues in the linker is not particularly limited, but can be defined as 250 residues, preferably up to 200, and more preferably up to 150 amino acid residues. In a preferred embodiment, the length of the polypeptide linker is 40 to 90 amino acids, preferably 50 to 80 amino acids, and more preferably 60 to 70 amino acids. In a specific embodiment, the polypeptide linker consists of 61 amino acids.

[0118] Site-specific endonucleases provide the proteins of the present invention, optionally binding to other components, and have the ability to detect target sites on endogenous DNA of eukaryotic cells or eukaryotic organisms to guide the proteins of the present invention (e.g., fusion proteins) including exonucleases to the target sites and cleave the endogenous DNA at the target sites. The term "target site" refers to a site on endogenous DNA that is to be cleaved by an endonuclease. The endonuclease of the proteins of the present invention (and the endonuclease domain of the fusion proteins) has site-specific endonuclease function and can cleave DNA at the target sites. In a preferred embodiment, double-strand breaks (DSBs) are induced in the endogenous DNA. The DSB can be a blunt-ended DSB or a staggered DSB with sticky overhangs. The type of cleavage at the target site depends on the endonuclease used. Some endonucleases, such as Cas9, induce blunt-ended DSBs in DNA, while others, such as Cas12a (formerly known as Cpf1), induce staggered DSBs with sticky overhangs. Endonucleases with nicking activity, i.e., so-called nickases, can also be used. Such nickases can be mutant variants of CRISPR nucleases such as Cas9. In one embodiment, the nickase is used to induce DSB in an endogenous DNA molecule, wherein the nickase induces a single-strand nick at both the adjacent coding and template strands. Two gRNAs can be used to guide the nickase to two sites to generate DSB through two nickase reactions.

[0119] 5'-3' exonucleases, through their 5'-3' exonuclease activity, provide the proteins of this invention with the ability to process DNA at target sites after endonuclease cleavage. Fusion or other binding with endonucleases ensures that the exonucleases are close to the DNA ends produced by the endonucleases. The inventors anticipate that the 5'-3' exonucleases have a higher affinity for the ends of double-stranded DNA than DNA repair factors of the NHEJ pathway, preventing the latter from binding to the DNA ends. This inhibition of the NHEJ pathway is thought to promote DNA break repair via the HDR pathway, thereby increasing the frequency of gene substitution events. The inventors also anticipate that differences in affinity for broken DNA ends will lead to the observation that some 5'-3' exonucleases produce higher gene substitution efficiencies than others. It is conceivable that the high activity of the exonucleases used in this invention against the DSB produced by the endonucleases effectively competes with the NHEJ pathway.

[0120] After binding to the DNA break ends, the exonuclease processes the DNA in a 5' to 3' orientation, preferably generating free 3'-overhangs at both ends of the cleaved DNA. The inventors hypothesize that this processing facilitates DNA break repair via the homology-mediated repair (HDR) pathway rather than the non-homologous end joining (NHEJ) pathway, particularly when suitable donor DNA is available (further details below). The 3' overhang is thought to pair with the complementary strand of the homologous arm of the donor nucleic acid (preferably donor DNA) to generate a hybrid DNA complex containing the cleaved endogenous DNA and the donor nucleic acid. It is also thought that the formation of this complex contributes to increasing the frequency of DNA break repair via the homology-mediated repair (HDR) pathway rather than the frequency of DNA break repair via the non-homologous end joining (NHEJ) pathway.

[0121] The endonucleases and exonucleases contained in the proteins of this invention are described in further detail below.

[0122] Site-specific endonucleases and their optional other components

[0123] The site-specific endonuclease for the fusion protein can be any endonuclease that cleaves double-stranded DNA at the target site in a site-specific manner. Examples of site-specific endonucleases that can be used in this invention are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-endonucleases, the latter being preferred due to their ease of use and wide applicability. Examples of CRISPR-endonucleases are Cas9 and Cas12a (formerly known as Cpf1) and their modified forms (e.g., mutants) having endonuclease activity. The structure and use of CRISPR-endonucleases Cas9 are described in particular in WO2014093712A1 and WO2014093635A1. The structure and use of CRISPR-endonucleases Cpf1 are described in particular in WO2016205711A1 and WO2017141173A1. In a preferred embodiment, the site-specific endonuclease is Cas9 or a mutant thereof having endonuclease activity.

[0124] It is generally known that CRISPR-based endonucleases such as Cas9 require a guide RNA (gRNA) to guide the endonuclease to the target site via complementarity with a sequence on the target DNA. Therefore, the gRNA is complementary to the target nucleic acid (typically the target DNA) and has the ability to bind to an endonuclease for cleaving the target DNA. As mentioned above, the nuclease can be Cas9 or Cpf1, or a modified form of it (e.g., a mutant) with endonuclease activity. However, the invention is not limited to Cas9 or Cpf1 endonucleases, and other CRISPR endonucleases can also be used. In some embodiments, the gRNA contains a guide sequence linked to a direct repeat sequence. The guide sequence provides complementarity with the target DNA to guide the endonuclease to the target site. The direct repeat sequence typically provides a portion that allows the gRNA to bind to a CRISPR nuclease (e.g., in tracrRNA). gRNA can be a single guide RNA (sgRNA), meaning it can contain the trans-activating RNA (tracrRNA) required by certain CRISPR-Cas systems (e.g., the type II CRISPR-Cas9 system). Therefore, gRNA can contain a sequence complementary to the target DNA and, if desired, the trans-activating CRISPR RNA (tracrRNA). The sequence complementary to the target DNA can have a length of 19 to 22 consecutive nucleotides, preferably 20 to 21 nucleotides. The continuity of these elements depends on the type of CRISPR-Cas system used.

[0125] For the use of another endonuclease in the Cas9 or type II CRISPR-Cas system, the gRNA typically contains a sequence complementary to the target DNA strand in the 5' to 3' direction and an sgRNA that transactivates the CRISPR RNA (tracrRNA). The use of the CRISPR-Cas system is generally known to those skilled in the art.

[0126] To specifically cleave target DNA, CRISPR endonucleases (also simply CRISPR nucleases) such as Cas9, which bind to gRNA (e.g., sgRNA), can scan endogenous DNA in eukaryotic cells to recognize target sequences adjacent to the proto-spacer adjacent motif (PAM-sequence) at the target site. When the PAM-sequence is detected at the target site, the endonuclease binds to it and unwinds the DNA. Subsequently, the distal portion of the gRNA bound to the endonuclease hybridizes with the unwound target DNA to identify the target site determined by the gRNA. Once approximately 20 consecutive nucleotides from the distal end of the gRNA have successfully hybridized with the separated DNA strand, the endonuclease can function and cleave the target DNA near the PAM sequence or nick it. The pattern of DNA cleavage depends on the nature of the endonuclease. CRISPR nucleases typically introduce double-strand breaks (DSBs). DSBs can have blunt ends (e.g., in the case of Cas9). If a DSB with sticky ends is desired, Cpf1 can be used as a CRISPR nuclease. Alternatively, the target DNA can be nicked, i.e., only one strand of the target DNA can be cleaved. Nicking can be achieved by using a CRISPR nuclease having one of the two nuclease domains of a native CRISPR nuclease that has been inactivated by mutation. However, in this invention, it is preferable to cleave both strands of the target DNA to introduce a DSB into the target DNA. Even more preferably, both strands of the target DNA are cleaved to introduce a blunt-ended DSB into the target DNA. In another embodiment, both strands of the target DNA are cleaved to introduce a sticky-ended DSB into the target DNA.

[0127] CRISPR nucleases are classified into different types based on their mode of operation. They originate from different bacteria and / or archaea and differ in size, domain structure, and the PAM-sequences they recognize. However, CRISPR / Cas nucleases rely on the fundamental principle of RNA-guided nuclease activity. Cpf1, as an example of a CRISPR nuclease, differs from Cas9 in that it recognizes different PAM-sequences and does not require the tracrRNA sequence in the gRNA (EP 3 009 511; Zetsche et al., Cell 163(3)(2015)759-771). Unlike Cas9, Cpf1 produces double-strand breaks with sticky overhangs.

[0128] Site-specific endonucleases may optionally possess nicking enzyme activity and introduce single-strand breaks (nicks) into the endogenous DNA of eukaryotic or prokaryotic cells. Single-stranded nicks are required at both the coding and template strands to generate a DSB (disseminated substrate nick) in the DNA using a nicking enzyme. Both nicks can be made by the same nicking enzyme. Alternatively, two different guide gRNAs, one targeting the coding strand and the other the template strand, can be used to obtain the DSB. In one embodiment, two gRNAs guide the nicking enzyme to introduce single-stranded nicks at both the coding and template strands of adjacent DNA, with one guide RNA designed in a PAM-in direction and the other in a PAM-out direction.

[0129] 5'-3' exonuclease

[0130] The exonucleases of the proteins (e.g., fusion proteins) of this invention are typically monomeric 5'-3' exonucleases. An exonuclease is a monomeric 5'-3' exonuclease in the sense of this invention if it consists of a single protein subunit and the exonuclease activity is present in that single protein subunit. Typically, many 5'-3' exonucleases known in the art are polymeric. However, the inventors have surprisingly discovered that if the exonuclease is a monomeric exonuclease, far better results and better gene editing efficiency can be achieved, especially when using the HDR mechanism.

[0131] If the exonuclease hydrolyzes blunt-terminated double-stranded DNA from both ends of the DSB in a 5' to 3' orientation to produce an unhydrolyzed 3'-spike, then it contains 5'-3' exonuclease activity. However, the activity is not limited to hydrolyzing blunt-terminated double-stranded DNA. In one embodiment of the invention, the 5'-3' exonuclease has a concentration of at least 0.072 μM·s when measured using the exonucleases described herein. -1 The catalytic efficiency k of 5'-3' exonuclease cat / K m Or at least 0.50s -1The 5'-3' exonuclease has a transformation number of at least 0.10, preferably at least 0.20 (μM·s). In a more preferred embodiment, the 5'-3' exonuclease has a transformation number of at least 0.10, preferably at least 0.20 (μM·s). -1 The catalytic efficiency k of 5'-3' exonuclease cat / K m , and / or at least 0.70s -1 Preferably at least 1.4s -1 The 5'-3' exonuclease with the transition number. Catalytic efficiency and / or transition number can be determined using the hairpin oligonucleotide of SEQ ID NO: 37 as a substrate, which is phosphorylated at its 5' end and carries a fluorescent dye linked to the thymine base (T) closest to the 3'-end of the oligonucleotide. The basic assay is described in Example 4. The assay was performed at 27°C and pH 7.9 (measured at 25°C), and the increase in fluorescence due to reduced fluorescence quenching was monitored, recorded, and plotted. The initial rate using multiple different substrate concentrations was determined from the initial linear portion of the graph. Transition number and K m The values ​​were determined by a Lineweaver-Burk plot of initial velocity versus substrate concentration. Example 4 provides further details regarding the determination.

[0132] In this document, when referring to the turn number and / or catalytic efficiency of exonucleases, these values ​​refer to free exonucleases and are determined using free exonucleases, which are exonucleases not part of the protein of this invention. For the purposes of this invention, it is assumed that the turn number and catalytic efficiency of free exonucleases correspond to the turn number and catalytic efficiency of fusion proteins or protein complexes containing exonucleases.

[0133] In another embodiment, in the in vitro exonuclease assay described in Example 4, the 5'-3' exonuclease of the present invention exhibits a higher 5'-3' exonuclease activity than the T5 exonuclease (SEQ ID NO: 31). Preferably, in the in vitro exonuclease assay described in Example 4, the 5'-3' exonuclease of the present invention exhibits at least twice the 5'-3' exonuclease activity of the T5 exonuclease (SEQ ID NO: 31), more preferably at least three times, and more preferably at least four times.

[0134] In another embodiment, in the in vitro exonuclease assay described in Example 4, the 5'-3' exonuclease of the present invention has the same or higher 5'-3' exonuclease activity as the phage T7 exonuclease (SEQ ID NO: 30).

[0135] The inventors have surprisingly discovered that the efficiency of gene editing using the HDR mechanism depends on the exonuclease activity. Furthermore, there are no particular limitations on the exonuclease, except that it is a monomeric 5'-3' exonuclease. Natural exonucleases can be used for fusion with endonucleases. However, natural exonucleases can be modified, for example by introducing mutations, additions, insertions, and / or deletions, provided that the exonuclease activity is not impaired. Examples of exonucleases are T7 exonuclease (SEQ ID NO: 30), UL12-1 exonuclease (SEQ ID NO: 32), and UL12-2 exonuclease (SEQ ID NO: 33). Further examples are BGLF5 exonuclease (SEQ ID NO: 34), DUMAS exonuclease (SEQ ID NO: 35), and MD5 exonuclease (SEQ ID NO: 36). Preferred exonucleases are PapE (SEQ ID NO: 43), a deoxyribonuclease derived from Papiine α-herpesvirus 2; and PiE (SEQ ID NO: 44), a deoxyribonuclease derived from Pteropus lylei-associated α-herpesvirus. Further examples of exonucleases are SOX (SEA ID NO: 68) and AB4P (SEQ ID NO: 69). Other particularly suitable exonucleases are ME15 (SEQ ID NO: 45) and SpiPh (SEQ ID NO: 46). Other suitable exonucleases are O3-12 (SEQ ID NO: 70), PhBO2 (SEQ ID NO: 71), and RaTL1 (SEQ ID NO: 72). Variants of these exonucleases as defined herein are also suitable for carrying out the invention.

[0136] The 5'-3' exonucleases that can be used in this invention can be divided into the following two groups: (I) UL-12 homologs (some of which are in Figure 15 (shown in) and (II) T7 homologs (some of which are in) Figure 16 (as shown in the image).

[0137] Members and examples of group (I) include: UL12-1 exonuclease (SEQ ID NO: 32), UL12-2 exonuclease (SEQ ID NO: 33), BGLF5 exonuclease (SEQ ID NO: 34), DUMAS exonuclease (SEQ ID NO: 35), MD5 exonuclease (SEQ ID NO: 36), PapE exonuclease (SEQ ID NO: 43), PiE exonuclease (SEQ ID NO: 44), SOX exonuclease (SEA ID NO: 68), AB4P exonuclease (SEQ ID NO: 69); and variants of these exonucleases as defined herein. Preferably, are UL12-1 exonuclease (SEQ ID NO: 32), UL12-2 exonuclease (SEQ ID NO: 33), PapE exonuclease (SEQ ID NO: 43), and PiE exonuclease (SEQ ID NO: 44), and their variants. The most preferred are PapE exonuclease (SEQ ID NO: 43) and PiE exonuclease (SEQ ID NO: 44) and their variants.

[0138] Members and examples of group (II) include: T7 exonuclease (SEQ ID NO: 30), ME15 exonuclease (SEQ ID NO: 45), SpiPh exonuclease (SEQ ID NO: 46), O3-12 exonuclease (SEQ ID NO: 70), PhBO2 (SEQ ID NO: 71), and RaTL1 exonuclease (SEQ ID NO: 72), as well as variants thereof as defined herein. Preferred are ME15 exonuclease (SEQ ID NO: 45) and SpiPh exonuclease (SEQ ID NO: 46), and variants thereof as defined herein. Most preferred are ME15 exonuclease and its variants as defined below.

[0139] Various exonucleases used in this invention are described in further detail below. Variants of items (ii) through (iv) possess 5'-3'-exonuclease activity (as in item (i), preferably the minimum activities as defined numerically above. Throughout this specification, amino acid sequences are given using the standard single-letter code of WIPO Standard ST25. X represents any of the 20 standard amino acid residues.

[0140] exonuclease of group (I)

[0141] In one embodiment, the 5'-3' exonuclease of the present invention is a protein whose amino acid sequence comprises or is composed of the following:

[0142] (i) The amino acid sequence of SEQ ID NO: 32 (UL12-1 exonuclease) or SEQ ID NO: 33 (UL12-2 exonuclease); or

[0143] (ii) An amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33; or

[0144] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33; or

[0145] (iv) An amino acid sequence having 1 to 50, preferably up to 40, more preferably up to 30, even more preferably up to 20, and most preferably up to 10 amino acid substitutions, additions, deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33.

[0146] In another embodiment, the 5'-3' exonuclease of the present invention is a protein whose amino acid sequence comprises or is composed of the following:

[0147] (i) The amino acid sequence of SEQ ID NO: 34 (BGLF5 exonuclease); or

[0148] (ii) An amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 34; or

[0149] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 34; or

[0150] (iv) An amino acid sequence having 1 to 50, preferably up to 40, more preferably up to 30, even more preferably up to 20, and most preferably up to 10 amino acid substitutions, additions, deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO: 34.

[0151] In another embodiment, the 5'-3' exonuclease of the present invention is a protein whose amino acid sequence comprises or is composed of the following:

[0152] (i) The amino acid sequence of SEQ ID NO: 35 (DUMAS exonuclease); or

[0153] (ii) An amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 35; or

[0154] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 35; or

[0155] (iv) An amino acid sequence having 1 to 50, preferably up to 40, more preferably up to 30, even more preferably up to 20, and most preferably up to 10 amino acid substitutions, additions, deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO: 35.

[0156] In another embodiment, the 5'-3' exonuclease of the present invention is a protein whose amino acid sequence comprises or is composed of the following:

[0157] (i) The amino acid sequence of SEQ ID NO: 36 (MD5 exonuclease); or

[0158] (ii) An amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 36; or

[0159] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 36; or

[0160] (iv) An amino acid sequence having 1 to 50, preferably up to 40, more preferably up to 30, even more preferably up to 20, and most preferably up to 10 amino acid substitutions, additions, deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO: 36.

[0161] The variants of items (ii) to (iv) above have 5'-3'-exonuclease activity, preferably those defined numerically above.

[0162] As described above, in a preferred embodiment, the 5'-3' exonuclease is a protein with the following amino acid sequence or containing the following amino acid sequence:

[0163] (i) The amino acid sequence defined in SEQ ID NO: 43 (PapE), or

[0164] (ii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence identity with the amino acid sequence defined in SEQ ID NO: 43, or

[0165] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably 95%, and most preferably 98% sequence similarity to the amino acid sequence defined in SEQ ID NO: 43, or

[0166] (iv) An amino acid sequence with 1 to 121 amino acid substitutions, additions, insertions and / or deletions, more preferably 1 to 90 amino acid substitutions, more preferably 1 to 60 amino acid substitutions, even more preferably 1 to 45 amino acid substitutions, and most preferably 1 to 30 amino acid substitutions compared to the amino acid sequence defined in SEQ ID NO: 43.

[0167] The amino acid sequences of the 5'-3' exonucleases in this embodiment (especially items (ii) to (iv)) preferably contain the amino acid sequences PAASVH, RRL, and APASAPAAVRAA (SEQ ID NO: 50) at those positions corresponding to PapE (see Figure 27). PapE-group specific motif I And / or II and / or III (see Figure 27, underlined to indicate motifs identified in Figure 27). More preferably, all three motifs are present.

[0168] Optionally or additionally, the amino acid sequence of the 5'-3' exonuclease (particularly items (ii) to (iv)) of this embodiment preferably comprises one or more amino acid sequence segments selected from SEQ ID NO:47 (APAESVHACGVL), SEQ ID NO:48 (APAASVHACGVL), SEQ ID NO:49 (AKYAFDPADAGXXVVAAHRRL), SEQ ID NO:50 (APASAPAAVRAA), and SEQ ID NO:51 (LIITPVRXDAA), more preferably at positions corresponding to those in SEQ ID NO:43.

[0169] In another preferred embodiment, the 5'-3' exonuclease of the present invention is a protein with the following amino acid sequence or containing the following amino acid sequence:

[0170] (i) The amino acid sequence defined in SEQ ID NO: 44 (PiE), or

[0171] (ii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence identity with the amino acid sequence defined in SEQ ID NO: 44, or

[0172] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably 95%, and most preferably 98% sequence similarity to the amino acid sequence defined in SEQ ID NO: 44, or

[0173] (iv) An amino acid sequence with 1 to 131, preferably 1 to 98, more preferably 1 to 65, even more preferably 1 to 49, and most preferably 1 to 32 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence defined in SEQ ID NO: 44.

[0174] The exonucleases of group (i), particularly PapE and PiE and their variants as defined above as SEQ ID NO: 43 and 44, can be proteins whose amino acid sequences include segments of the amino acid sequence SEQ ID NO: 54 (FRYCVGRAD) and / or SEQ ID NO: 55 (PXPLMXFFEAATQ), for example at positions corresponding to those in SEQ ID NO: 43.

[0175] Preferred implementation scheme of exonuclease for group (I)

[0176] Group (I) exonucleases typically share certain sequence motifs as shown in the alignment in Figure 27. Variants of the exonucleases given above preferably possess these sequence motifs, such as the amino acid sequence K / RPLMXFF / YE. UL12-Group Special Heterogeneous motif I In this amino acid sequence segment, K / R represents K or R, and F / Y represents F or Y. X is as defined above.

[0177] As an alternative or supplement to the above implementation scheme, group (I) exonuclease is a group containing UL12-Group Specific Motif I , Motif I , bridge district, motif Ia and UL12-Group Specific Motif IIThe 5'-3' exonuclease is shown in Figure 27 (the underlined section highlights the segment shown in Figure 27). Therefore, the 5'-3' exonuclease can be an exonuclease whose amino acid sequence contains the segment of SEQ ID NO: 56 (PXPLMXFXEAATQXQXXXQLWXLLRRGLXTAXTLXWGXXGPXFXXXWLXXXXXXXXXXXXXAXXFGRXNEXXARXXLFRYCVGRAD), wherein the X at position 35 of this sequence is preferably K or R. This segment is preferably located at the position corresponding to the position in UL12-1.

[0178] Alternatively, group (I) exonuclease may contain Motif I Referring to Figure 27. Therefore, the 5'-3' exonuclease can be an amino acid sequence segment containing amino acid residues 9 to 37 of SEQ ID NO: 56, and contains an amino acid sequence segment containing amino acid residues 9 to 37 of SEQ ID NO: 56, wherein the X at position 35 of SEQ ID NO: 56 is preferably K or R.

[0179] As an alternative or supplement to the above implementation scheme, the exonuclease of group (I) is a nuclease containing... UL12-Group Specific Motif II The 5'-3' exonuclease, see Figure 27. The 5'-3' can therefore be an exonuclease whose amino acid sequence contains the amino acid sequence segment RYCV or FRYCV, preferably followed by the segment GRAD, producing segment RYCVGRAD or FRYCVGRAD, respectively. As mentioned above, such sequence segments are preferably located at positions corresponding to those in UL12-1.

[0180] Optionally or additionally, the exonuclease of group (I) may contain Motif II Referring to Figure 27. Therefore, the 5'-3' exonuclease can be a protein whose amino acid sequence contains the amino acid sequence segment of SEQ ID NO: 95 (GVLXDXHTGMVGASLD), wherein the X at position 4 is preferably M, V, L or I; the H at position 7 can optionally be R; and / or the M at position 10 can optionally be V or L.

[0181] Optionally or additionally, the exonuclease of group (I) may contain Motif III See Figure 27. Therefore, the 5'-3' exonuclease can be a protein whose amino acid sequence includes the amino acid sequence segment of SEQ ID NO: 96 (EVKCRAKYAFDPXD), wherein the V at position 2 can optionally be I; the A at position 9 can optionally be L or T; and / or the D at position 14 can optionally be E.

[0182] Optionally or additionally, the exonuclease of group (I) may contain Sequence VI Referring to Figure 27. Therefore, the 5'-3' exonuclease can be a protein whose amino acid sequence includes the amino acid sequence segment of SEQ ID NO: 97 (FANPRHPNFKQILVQXYVLXXHFP), wherein the K at position 10 can optionally be R; and / or the X at position 16 is preferably G, A, S or T.

[0183] Other preferred embodiments including the 5'-3' exonuclease UL12 and variants

[0184] In a preferred embodiment, the protein used for editing endogenous DNA is a fusion protein comprising a CRISPR-nuclease as an endonuclease and UL12 (UL12_1 or UL12_2 or variants thereof as defined herein) as an exonuclease. In this embodiment, the protein used for editing endogenous DNA is preferably a protein.

[0185] The site-specific endonucleases mentioned above are CRISPR-Nucleases as defined above, and

[0186] The site-specific endonuclease and the 5'-3' exonuclease are fused via a polypeptide linker, wherein the polypeptide linker is 25 amino acids or more in length, preferably 30 amino acids or more, more preferably 40 amino acids or more, even more preferably 50 amino acids or more, and most preferably 60 amino acids or more.

[0187] The 5'-3' exonuclease is a protein with the following amino acid sequence or containing the following amino acid sequence:

[0188] (i) The amino acid sequence defined in SEQ ID NO: 32 (UL12-1) or SEQ ID NO: 33 (UL12-2), or

[0189] (ii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence identity with the amino acid sequence defined in SEQ ID NO: 32 or SEQ ID NO: 33, or

[0190] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence defined in SEQ ID NO: 32 or SEQ ID NO: 33, or

[0191] (iv) An amino acid sequence with 1 to 120 amino acid substitutions, additions, insertions and / or deletions, more preferably 1 to 93 amino acids, even more preferably 1 to 62 amino acids, and even more preferably 1 to 46 amino acids, and most preferably 1 to 31 amino acid substitutions, additions, insertions and / or deletions, compared to the amino acid sequence defined in SEQ ID NO: 32 or SEQ ID NO: 33.

[0192] This implementation can be combined with other preferred implementations described herein, such as those containing the amino acid sequence segment of SEQ ID NO: 56.

[0193] exonuclease of group (II)

[0194] In another preferred embodiment, the 5'-3' exonuclease of the present invention is a protein whose amino acid sequence comprises or is composed of the following:

[0195] (i) The amino acid sequence of SEQ ID NO: 30 (T7 exonuclease); or

[0196] (ii) An amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 30; or

[0197] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 95%, even more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 30; or

[0198] (iv) An amino acid sequence having 1 to 50, preferably up to 40, more preferably up to 30, even more preferably up to 20, and most preferably up to 10 amino acid substitutions, additions, deletions, and / or insertions compared to the amino acid sequence of SEQ ID NO: 30.

[0199] In a preferred embodiment, the 5'-3' exonuclease of the present invention is a protein with the following amino acid sequence or containing the following amino acid sequence:

[0200] (i) The amino acid sequence defined in SEQ ID NO: 45 (ME15), or

[0201] (ii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence identity with the amino acid sequence defined in SEQ ID NO: 45, or

[0202] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence defined in SEQ ID NO: 45, or

[0203] (iv) An amino acid sequence comprising, substituted, added, inserted and / or deleted amino acids, of 1 to 60, preferably 1 to 45, more preferably 1 to 30, even more preferably 1 to 22, and most preferably 1 to 15 amino acids, compared to the amino acid sequence defined in SEQ ID NO: 45.

[0204] Optionally, the amino acid sequence of the 5'-3' exonuclease in this embodiment may include one or more amino acid sequence segments selected from SEQ ID NO:52 (APTESETLWDCI) and SEQ ID NO:53 (ILRFNDYNIDT).

[0205] In another preferred embodiment, the 5'-3' exonuclease of the present invention is a protein with the following amino acid sequence or containing the following amino acid sequence:

[0206] (i) The amino acid sequence defined in SEQ ID NO: 46 (SpiPh), or

[0207] (ii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence identity with the amino acid sequence defined in SEQ ID NO: 46, or

[0208] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence defined in SEQ ID NO: 46, or

[0209] (iv) An amino acid sequence with 1 to 59 amino acid substitutions, additions, insertions and / or deletions, more preferably 1 to 44 amino acid substitutions, more preferably 1 to 29 amino acid substitutions, even more preferably 1 to 22 amino acid substitutions, and most preferably 1 to 14 amino acid substitutions compared to the amino acid sequence defined in SEQ ID NO: 46.

[0210] The 3'-spikes produced by the action of exonucleases (preferably at both ends of the cleaved endogenous DNA) are homologous to the homologous arms of the donor nucleic acid and can anneal with them. This annealing is the starting point for DNA break repair via HDR.

[0211] When a protein is defined herein by the number or range of amino acid substitutions, additions, deletions, and / or insertions, these substitutions, additions, deletions, and insertions can be combined, but the given number or range refers to the sum of all substitutions, additions, insertions, and deletions of amino acid residues compared to the reference sequence defined by SEQ ID NO. Among the amino acid substitutions, additions, insertions, and deletions, amino acid substitutions, additions, and deletions are preferred. The term "insertion" refers to the insertion of an amino acid residue within the amino acid sequence of the reference sequence, i.e., an addition excluding the C- or N-terminus. The term "addition" refers to the addition of an amino acid residue at the C- or N-terminus of the amino acid sequence of the reference sequence. "Deletion" can be the deletion of an amino acid residue at the end or within the reference sequence. In this document, when a protein or its domains are defined by the number or range of amino acid substitutions, additions, deletions, and / or insertions relative to the reference sequence, as an alternative embodiment, the protein may have one to several amino acid substitutions, additions, insertions, or deletions relative to the indicated amino acid sequence of the segment.

[0212] donor nucleic acid

[0213] The donor nucleic acid (sometimes also referred to as a "repair template," "donor fragment," or, if it is DNA, a "DNA repair template" or "donor DNA") used with the protein of the present invention (i.e., the fusion protein or oligomeric protein (protein complex) of the present invention) is a nucleic acid molecule that typically contains a donor sequence flanked by first and second homologous arms, one at the 5' end of the donor nucleic acid and the other at the 3' end of the donor nucleic acid. The first homologous arm is typically homologous to a first region flanking the target site on the first side of the target site in the cell genome. The second homologous arm is homologous to a second region flanking the target site on the second side of the target site.

[0214] When provided to cells, the donor nucleic acid can be single-stranded or double-stranded DNA or RNA, and can be linear or circular. However, if the donor nucleic acid is provided to cells as RNA, it typically needs to be transcribed from RNA into donor DNA within the cells for HDR to function. The donor nucleic acid can be transcribed from RNA into DNA by reverse transcriptase, and can also be provided to cells in addition to donor RNA. To avoid reverse transcription, the donor nucleic acid is preferably DNA, and in this embodiment, it is also referred to herein as donor DNA.

[0215] The donor nucleic acid can be part of a DNA construct, plasmid, or vector. The homologous arms of the donor nucleic acid contain nucleotide sequences homologous to endogenous DNA near the target site. The 5' homologous arm of the donor nucleic acid can be homologous to nucleotide sequences upstream of the target site, and the 3' homologous arm can be homologous to nucleotide sequences downstream of the target site. Due to this homology, a 3' overhang generated by the 5'-3' exonuclease of the protein of this invention can invade the homologous arms of the donor nucleic acid and anneal to the complementary strand of the homologous arm. This results in a hybrid DNA complex containing endogenous DNA and the donor nucleic acid. This hybrid DNA complex is also called a "displacement loop" (D-loop), representing the first step in DNA DSB repair via HDR. To untangle the displacement loop, the 3' overhang annealed to the homologous arm can be used as a primer for a DNA polymerase to synthesize a new DNA strand using the homologous arm as a template. This method allows a copy of the donor sequence contained in the donor nucleic acid to be inserted into endogenous DNA located at the target site. Specific nucleotide sequences can be deleted at the target site in the endogenous DNA. For this purpose, a donor sequence shorter than the nucleotide sequence it replaces in endogenous DNA can be used, with appropriate selection of homologous arms. The donor sequence may even be absent in cases of deletion of nucleotide sequences (e.g., a nonspecific number of nucleotides) in endogenous DNA.

[0216] In one embodiment, the protein of the present invention is combined with a donor nucleic acid and delivered to a eukaryotic cell for editing endogenous DNA using HDR. In an alternative embodiment, the protein of the present invention is delivered to the cell without a donor nucleic acid. In this embodiment, the protein of the present invention can produce a deletion of at least one nucleotide in endogenous DNA in the immediate vicinity of a double-strand break generated at the target site. Preferably, this embodiment produces a deletion of two or more consecutive nucleotides in the immediate vicinity of a double-strand break generated at the target site. Such an embodiment is particularly useful for randomly deleting one or more nucleotides in the non-coding regions of endogenous DNA.

[0217] Donor nucleic acids can be introduced into cells in many different ways known to those skilled in the art. Depending on the delivery method, the donor nucleic acid can be introduced into the cell in the form of single-stranded or double-stranded DNA or RNA. In the case of plants, the donor nucleic acid can be introduced into cells via Agrobacterium-mediated transformation. To provide the donor nucleic acid into plant cells or plant cells, the cells or plant are brought into contact with an Agrobacterium cell suspension carrying the donor nucleic acid within T-DNA in a T-DNA binary plasmid. According to current knowledge, Agrobacterium cells secrete T-DNA into plant cells as single-stranded DNA, which, as demonstrated in the following examples, is sufficient to enable HDR to function and ultimately lead to the gene replacement (gene targeting) described in this invention. However, when secreted into plant cells as part of single-stranded T-DNA, the donor nucleic acid may also be converted into double-stranded DNA before HDR occurs. In either case, providing the donor nucleic acid into plant cells in single-stranded or double-stranded form using Agrobacterium transformation triggers HDR and ultimately leads to the gene replacement or editing described in this invention.

[0218] As described above, alternatively, the donor nucleic acid can be provided to the cell in the form of RNA, for example, via delivery by an RNA virus or after expression from a transgene. However, if the donor nucleic acid is provided to the cell in the form of RNA, it should be reverse transcribed into DNA using a reverse transcriptase that can be co-expressed within the cell.

[0219] Other transformation or transfection methods commonly known in the art can be used. For example, donor nucleic acids can also be delivered into eukaryotic cells, particularly animal cells, by electroporation, chemical transfer using transfection reagents, or by gene bombardment.

[0220] The donor nucleic acid can be linear single-stranded DNA. However, preferably, the donor nucleic acid is linear double-stranded DNA, more preferably linear double-stranded DNA. In any case, the donor nucleic acid contains a first homologous arm and a second homologous arm as described above. The donor nucleic acid may optionally contain a donor sequence located between the two homologous arms. The first homologous arm may be located at the 5' end of the donor nucleic acid and contains a nucleotide sequence that preferably shares at least 95% sequence identity with a fragment of endogenous DNA located upstream of the target site. The second homologous arm at the 3' end of the donor nucleic acid contains a nucleotide sequence that shares at least 95% sequence identity with a fragment of endogenous DNA located downstream of the target site. In a preferred embodiment, the homologous arms of the donor nucleic acid show no mismatch with the endogenous DNA. In another preferred embodiment, the homologous arms perfectly match the endogenous DNA at least in the 5' and 3' directions of the donor nucleic acid molecule. If the homologous arm shares at least 95% sequence identity with a segment of endogenous DNA, the nucleotide sequence length of the homologous arm shall be at least 20 bp, preferably at least 60 bp, more preferably at least 60 bp, and most preferably at least 120 bp, and the sequence segment with the highest identity shall be oriented toward the 5' and 3' ends of the donor nucleic acid molecule.

[0221] Each homologous arm should have a minimum length of 20 bp and can be up to 1000 bp, preferably up to 500 bp, and more preferably up to 250 bp. Preferably, the length of the homologous arm is greater than 50 bp. In one specific embodiment, the length of each of the two homologous arms is between 100 and 300 bp, and allows for perfect pairing with endogenous DNA without mismatch.

[0222] The donor sequence (also referred to herein as the donor nucleotide sequence), optionally present in the donor nucleic acid, is a nucleotide sequence that can be inserted into the endogenous DNA at the target site. If the donor sequence is not present in the donor nucleic acid, no nucleotide sequence is to be integrated from the donor nucleic acid into the endogenous DNA. However, sequence segments can be deleted from the endogenous DNA, for example, sequences located between two homologous arms after the donor nucleic acid has been annealed with the endogenous DNA.

[0223] The donor sequence (if present) contains at least one nucleotide, preferably at least 10 nucleotides, more preferably at least 30 nucleotides, and even more preferably at least 100 nucleotides. There is no particular limitation on the maximum length of the donor sequence. The donor sequence can be up to 15,000 nucleotides long. In another embodiment, the donor sequence can also be 20,000 nucleotides (20 kb) long. However, the likelihood of a long donor sequence being inserted into endogenous DNA at the target site may be lower than that of a shorter donor sequence. In one embodiment, the donor sequence for repair template is up to 10,000 nucleotides long, preferably up to 7,000 nucleotides long, and more preferably up to 3,000 nucleotides long.

[0224] The inventors have discovered that the protein of this invention allows for the insertion of unexpectedly long donor nucleotide sequences contained in donor nucleic acids into endogenous DNA via HDR. Compared to the absence of exonucleases, the protein of this invention can increase the frequency of gene substitution events, preferably by several orders of magnitude, thereby enabling the insertion of unexpectedly long nucleotide sequences into endogenous DNA. The donor sequence may be or may contain one or more open reading frames (ORFs) or the complete gene to be inserted into endogenous DNA at the target site.

[0225] Methods of modifying or editing endogenous DNA

[0226] The proteins of this invention can be used to modify (also “edit”) eukaryotic endogenous DNA at target sites. The term “editing endogenous DNA” refers to modifying endogenous DNA at a target site. The fundamental mechanism of this modification is considered to be homology-mediated repair (HDR). Modifications in endogenous DNA depend on donor nucleic acids and are selected from insertion into, deletion from, and substitution of endogenous DNA. Here, insertion refers to the insertion of at least one nucleotide from the donor nucleic acid into the endogenous DNA at the target site. Substitution refers to the replacement of at least one nucleotide (preferably a segment of two or more nucleotides) of the endogenous DNA at the target site with at least one different nucleotide or a segment of two or more nucleotides from the donor nucleic acid. Deletion refers to the deletion of at least one nucleotide (preferably a segment of two or more nucleotides) from the endogenous DNA at the target site. Insertion, substitution, or deletion of nucleotides in endogenous DNA can be achieved by appropriately designing the donor nucleic acid.

[0227] The endogenous DNA in eukaryotic cells can be the cell's genomic DNA, but any double-stranded DNA molecule contained within the cell (such as mitochondrial DNA, plastids, or others) can be edited. Endogenous DNA can be edited at a single target site or simultaneously at two or more target sites.

[0228] The editing of endogenous DNA in this invention can also be referred to as "gene replacement" or "gene targeting." Gene replacement in this invention includes editing endogenous DNA as defined above. Specifically, gene replacement also includes targeted gene knockout resulting from the targeted deletion of one or more nucleotide sequence segments or single base pairs in the endogenous DNA of eukaryotic cells. This needs to be distinguished from non-target gene knockout where the nature of the mutation cannot be predicted in advance.

[0229] The protein of the present invention can increase the frequency of double-strand break repair via the homology-mediated repair (HDR) pathway. The present invention can also increase the number of gene substitution events in eukaryotic cells, presumably because DNA break repair via HDR is a prerequisite for gene substitution. Compared to the individual action of exonucleases and endonucleases not fused together or forming protein complexes, the protein of the present invention can achieve a higher number (i.e., increased frequency) of gene substitution events when provided with donor nucleic acids. In a preferred embodiment, the protein of the present invention increases the number of gene substitution events by at least 1.5 times, more preferably at least 3 times, and even more preferably at least 5 times, compared to providing exonucleases and endonucleases alone (i.e., when provided with donor nucleic acids without fusion or forming protein complexes). Further, compared to fusion proteins or protein complexes containing T5 exonuclease as an exonuclease, the protein of the present invention increases the number of gene substitution events by at least 1.5 times, more preferably at least 2 times, and even more preferably at least 3 times. Compared with fusion proteins or protein complexes containing polynucleotide exonucleases, the proteins of the present invention can increase the number of gene substitution events by at least 1.5 times, more preferably at least 2 times, and even more preferably at least 3 times.

[0230] The inventors have discovered that monomeric 5'-3' exonucleases exhibiting high in vitro 5'-3' exonuclease activity, when included in fusion proteins or protein complexes along with site-specific endonucleases and provided with donor nucleic acids, are particularly well-suited to significantly increase the frequency of HDR and ultimately increase the frequency of gene substitution events.

[0231] The editing of endogenous DNA in this invention is performed in eukaryotic cells. Preferably, it occurs in at least one cell of a eukaryotic organism. However, more preferably, the editing is performed in multiple cells of a eukaryotic organism, such as multiple cells in one or more leaves of a plant. The editing typically occurs in parallel in two or more cells.

[0232] The editing of endogenous DNA in eukaryotic cells according to the present invention requires the introduction of the proteins and donor nucleic acids of the present invention into the same cells. Eukaryotic cells typically contain functional HDR pathways naturally, and therefore cellular genetic engineering to provide HDR pathways to cells is generally unnecessary. Optionally, other components that modify and / or enhance the frequency and / or efficiency of DNA break repair via homologous recombination (HDR) can be introduced into the cells. For example, components such as proteins Rad51 and / or Rad52 can be introduced into the cells to support DNA break repair via HDR. Alternatively, components that downregulate the NHEJ pathway can be introduced into the cells to facilitate DNA break repair via HDR.

[0233] After successful editing in eukaryotic cells, an organism can be obtained from the edited eukaryotic cells. For example, after editing embryonic animal cells, an animal containing edited endogenous DNA in all cells can be obtained. After editing the germ cells of an animal, the edited germ cells can be used to fertilize another germ cell, or the edited germ cells can be fertilized by another germ cell to obtain embryonic cells containing edited endogenous DNA. In a further alternative, animal somatic cells containing edited endogenous DNA can be generated and propagated. For example, such cells can express proteins or other factors that are not expressed in a suitable form in the starting cells. If the edited cells are applied to an organism lacking said protein or factor, an organism lacking said protein or factor can be provided. Therefore, the present invention provides a method for treating or preventing genetic defects in eukaryotes (e.g., humans or animals), the method comprising modifying the endogenous DNA of eukaryotic cells at an endogenous DNA target site (of the present invention), culturing the modified cells and / or progeny cells of the modified cells, and applying one or more modified cells to the eukaryote in need.

[0234] After successful editing in eukaryotic plant cells, plants can be regenerated from said cells, wherein said plants contain endogenous DNA in all their cells. This can produce new plant lines (e.g., new plant lines of crop plants) containing the edited endogenous DNA. Methods for regenerating plants from cells or tissues are generally known in the field of plant biotechnology. For example, plants can be regenerated from callus tissue using suitable culture media, as described, for example, in textbooks on plant biotechnology, such as Slater, Scott, and Fowler, Plant Biotechnology, second edition, Oxford University Press, 2008. Alternatively, the editing can be performed in germline cells of floral tissue, for example using the floral dip transformation method described, for example, by Clough and Bent (The Plant Journal (1998) 16(6), 735-743). Seeds produced from these flowers can contain the edited endogenous DNA and can be selected and, if desired, further cultured to produce plant lines containing the edited endogenous DNA.

[0235] Eukaryotic cells and organisms that can be edited using this invention

[0236] The eukaryotic cells that can be edited according to the present invention can be fungal (e.g., yeast) cells, plant cells, or animal cells, such as human cells. Preferably, the eukaryotic cells are plant cells. Therefore, the organisms of the present invention can be plant or animal organisms. In one embodiment, the present invention does not include humans and / or the present invention does not include methods for modifying the human reproductive system. In one embodiment, the endogenous DNA of a single cell of a eukaryotic organism is edited. In another embodiment, the endogenous DNA of multiple cells of a eukaryotic organism (particularly plants) is edited. In a further embodiment, the endogenous DNA of somatic cells of a eukaryotic organism (particularly plants) is edited. In another embodiment, the endogenous DNA of reproductive cells is edited such that the edited endogenous DNA is inherited by offspring. In another embodiment, the endogenous DNA of embryonic cells is edited such that the edited endogenous DNA is present in all cells of an organism developing from the edited embryonic cells.

[0237] In animal cells, human cells can be edited according to the present invention. However, cells from livestock animals are preferred.

[0238] The plants in which the editing of the present invention can be carried out are not particularly limited. The present invention can be applied to monocotyledonous and dicotyledonous plants that can be edited according to the present invention. Plant species used to carry out the present invention include, but are not limited to, representatives of dicotyledonous plants such as Leguminosae, Solanaceae, Chenopodiaceae, Compositae, Cucurbitaceae, Brassicaceae, and Scrophulariaceae, and representatives of monocotyledonous plants such as Poaceae, Musaceae, and Zingiberaceae. Both crop and non-crop plants can be used, with crop plants being preferred. Common crops preferably edited with the proteins of this invention include alfalfa, barley, beans, canola, cowpeas, cotton, corn, clover, lotus, lentils, lupine, maize, millet, oats, peas, peanuts, poplar, rice, rye, sweet clover, sunflower, sweet pea, soybean, sorghum, triticale, yam beans, velvet beans, vetch, wheat, wisteria, potato, banana, coffee, cacao, sugar beet, and nut plants.

[0239] By implementing the method of the present invention using such plants or cells of such plants, cells of these plants containing edited endogenous DNA can be produced. As described above, plants containing edited endogenous DNA in all cells of the plant can thus be produced.

[0240] The nucleic acid, DNA constructs, plasmids and vectors of the present invention

[0241] The present invention also provides a nucleic acid molecule comprising a polynucleotide encoding the protein of the present invention. This nucleic acid molecule is also referred to herein as a first nucleic acid molecule. The polynucleotide should be understood as the coding sequence of the protein of the present invention, such as the coding sequence of the fusion protein of the present invention or the coding sequence of a subunit of the protein of the present invention. If the protein of the present invention is an oligoprotein, the nucleic acid molecule may comprise two coding sequences: a first coding sequence encoding a first subunit and a second coding sequence encoding a second subunit. Optionally, the first and second subunits of the oligoprotein, such as a dimer, may be encoded on separate nucleic acid molecules.

[0242] The present invention also provides a first DNA construct comprising a polynucleotide encoding the protein of the present invention. The present invention also provides a plasmid or vector comprising the DNA construct. The nucleic acid molecule and DNA construct may contain additional genetic elements, such as genetic elements for expressing the protein in eukaryotic cells. Examples of such genetic elements are promoters that are active in cells or organisms and effectively linked to the polynucleotide, optional transcription enhancers, and / or transcription terminators. For Agrobacterium-mediated transformation, the left and right T-DNA boundary sequences may also be such genetic elements. The first nucleic acid molecule, plasmid, or vector may further comprise additional nucleic acid segments, such as a plasmid or vector backbone. The nucleic acid molecule, DNA construct, plasmid, or vector may be single-stranded or double-stranded, and may be circular or linear; preferably, they are double-stranded and circular.

[0243] In one embodiment, the first nucleic acid molecule may further comprise a donor nucleic acid. In another embodiment, the first nucleic acid molecule may further encode gRNA for use as a CRISPR nuclease, the endonuclease of the present invention. In a further embodiment, the first nucleic acid molecule comprises a donor nucleic acid and encodes gRNA for the CRISPR nuclease. After transfection into one or more eukaryotic cells, the donor nucleic acid can be cleaved from the nucleic acid molecule, for example, using a CRISPR nuclease and additional gRNA, said additional gRNA guiding the CRISPR nuclease to the appropriate cleavage site on the nucleic acid molecule to cleave the donor nucleic acid.

[0244] The first nucleic acid molecule, DNA construct, plasmid, or vector of the present invention can be generated by cloning the elements to be combined together. A convenient cloning method also used in the examples is Golden Gate (GG) cloning, which utilizes an IIS-type restriction enzyme for restriction seamless ligation, see WO 2008 / 095927 and WO2011154147.

[0245] The present invention also describes a second nucleic acid molecule. This second nucleic acid molecule is, or comprises, the donor nucleic acid of the present invention as described above. The present invention also provides a second DNA construct comprising the donor nucleic acid and optional additional elements, such as left and right T-DNA boundaries for Agrobacterium-mediated transformation. The second nucleic acid molecule may contain additional nucleic acid segments, such as plasmids or vector backbones. Those skilled in the art will understand that the donor nucleic acid may be contained in either the first or second nucleic acid molecule. When the second nucleic acid molecule is used in conjunction with the donor nucleic acid, it may further encode gRNA for use as a CRISPR nuclease, which is the endonuclease of the present invention.

[0246] This invention describes a third nucleic acid molecule that contains or encodes gRNA, such as sgRNA. If the third nucleic acid molecule is RNA, it contains gRNA. If the third nucleic acid molecule is DNA, it contains a polynucleotide encoding gRNA. For transcription in eukaryotic cells, if the third nucleic acid molecule is DNA, it may contain a (third) DNA construct containing a promoter efficiently linked to the polynucleotide encoding gRNA. For transfection in plant cells using Agrobacterium-mediated transfection, the third DNA construct may further contain left and right T-DNA boundaries. As described above, additional gRNA may be present on or encoded on the third nucleic acid molecule, for example, for cleaving donor nucleic acids from the same or another nucleic acid molecule.

[0247] Promoters used for expression in eukaryotic cells are generally known. For expression in plant cells and plants, promoters active in plant cells are used. The term "promoter active in plant cells" refers to a DNA sequence that controls (initiates) transcription in plant cells. This includes any promoters of plant origin, as well as any promoters of non-plant origin capable of directing transcription in plant cells, i.e., certain promoters of viral or bacterial origin, such as the 35S promoter of cauliflower mosaic virus (CaMV35S promoter) (Harpster et al. (1988) Mol Gen Genet. 212(1): 182-90), promoters 4 or 7 of subterranean clover virus (WO9606932), or T-DNA gene promoters, including cell cycle-specific promoters (Ferreira et al., (1994) Plant Cell 6: 1763-1774), tissue-specific or organ-specific promoters, including but not limited to seed-specific promoters (e.g., WO89 / 03887), oocyte-specific promoters (Steffen et al., (2007) Plant J. 51: 281-292; Sprunck et al., (2012) Science). 338:1093-1097), organ primordium-specific promoters (An et al. (1996) Plant Cell 8(1):15-30), stem-specific promoters (Keller et al. (1988) EMBO J.7(12):3625-3633), leaf-specific promoters (Hudspeth et al. (1989) Plant Mol Biol.12:579-589), mesophylline-specific promoters (such as photoinducible Rubisco promoters), root-specific promoters (Keller et al. (1989) Genes Dev.3:1639-1646), tuber-specific promoters (Keil et al. (1989) EMBO J.8(5):1323-1330), vascular tissue-specific promoters (Peleman ... Examples of suitable promoters include 84:359-369, stamen-selective promoters (WO 89 / 10396, WO 92 / 13956), and dehiscence zone-specific promoters (WO 97 / 13865). For transient expression, constitutive promoters, i.e., promoters not regulated by development, are preferred. However, constitutive promoters can be tissue-specific or organ-specific.Preferred promoters are those used in the following embodiments.

[0248] Delivery of proteins, donor DNA, and / or gRNA into eukaryotic cells according to the present invention

[0249] The editing of endogenous DNA in this invention requires the simultaneous presence of a protein, a donor nucleic acid, and (in the case of a CRISPR nuclease as an endonuclease) one or more gRNAs in the same cell. These elements are also referred to herein as components of the invention. This means that the components of the invention should be present simultaneously in the same cell and therefore can be provided to said cell in parallel or continuously. The components can be provided transiently or stably to eukaryotic cells, cells of organisms, or organisms. Transiently means that it is highly unlikely, and usually does not occur, to incorporate one or more nucleic acid molecules or portions thereof encoding or containing said components into the genome of a eukaryotic cell (e.g., because no selective pressure is applied to incorporate nucleic acid molecules into the genome of a eukaryotic cell or organism). For example, DNA plasmids serving as nucleic acid molecules of the invention can contain or encode one or more components of the invention (effectively linked to a promoter when needed, so that said one or more components can be expressed by the DNA plasmid within the cell).

[0250] Stable delivery of the components to eukaryotic cells, organisms, or other organisms refers to the incorporation of one or more nucleic acid molecules, or portions thereof, encoding or containing one or more of the components, into the genome of the eukaryotic cell or organism (e.g., by applying selection pressure and selecting cells or organisms where such incorporation has occurred, or by using Agrobacterium-mediated transformation). Agrobacterium-mediated transformation typically integrates T-DNA containing or encoding one or more of the components into the genome of the cell. The genome then contains or encodes the components contained or encoded in the T-DNA, such that the components can be expressed or excised via promoters effectively linked to them (e.g., in the case of donor nucleic acids). If plant somatic cells or plant cells are transformed with Agrobacterium, the components are typically not transferred to the progeny plants of the transformed plant. If reproductive plant cells or plant cells are transformed with Agrobacterium, the components can be transferred to progeny cells or organisms, such that the components or the coding sequences encoding the components are stably integrated into the genome in the progeny.

[0251] All components of this invention need not be provided to eukaryotic cells or organisms in the same manner, i.e., all stably or all transiently. Instead, one or more components may be provided stably, and one or more other components may be provided transiently. For example, a first nucleic acid molecule encoding a target protein may be stably provided to a eukaryotic cell or organism. Then, a second nucleic acid molecule comprising the donor nucleic acid and optionally a third nucleic acid molecule encoding one or more gRNAs may be transiently provided to a cell stably containing and expressing the protein of this invention.

[0252] It is advantageous to transiently provide the components of the invention to eukaryotic cells, organisms, or other organisms, since it is generally desirable to restrict genetic modifications to specific desired editing events. However, as described above, one or more components of the invention can be stably incorporated into eukaryotic cells, organisms, or other organisms, allowing them to be expressed and transiently providing other components. Any unwanted stable genetic modifications can then be removed from the transgenic plant by segregation and outcrossing.

[0253] Several possibilities exist for providing eukaryotic cells with the components of the invention for carrying out the methods of the invention, particularly transiently. For example, the components can be injected into eukaryotic cells or taken up by eukaryotic cells (e.g., during electroporation or PEG-mediated transformation) as a mixture of the protein of the invention, donor DNA (or DNA containing donor DNA). These methods can be carried out in animal cells or plant cells. However, typically, these components are provided to cells by genetic transformation or transient transfection of nucleic acid molecules encoding fusion proteins and gRNA, causing them to be expressed in transformed or transfected eukaryotic cells, and the donor DNA is also transformed or transfected into the cells.

[0254] Methods for introducing one or more nucleic acid molecules into animal cells are known to those skilled in the art, such as electroporation, microinjection, or the use of transfection agents (e.g., as described in WO2014056590 or WO2014053245). These methods are particularly suitable for transiently delivering the components to cells.

[0255] In addition, various methods for introducing one or more DNA molecules into plant cells, plant cells, or plants are known, examples of which include electroporation, PEG (polyethylene glycol) conversion, microinjection, particle bombardment, and the use of viral vectors. These methods are also particularly suitable for the transient delivery of the components to plant cells. However, the preferred method for introducing the DNA molecules of the present invention into plant cells or plant cells is Agrobacterium-mediated transformation. Agrobacterium-mediated transformation is well-established in the field of plant biotechnology, for example, from textbooks on plant biotechnology, such as Slater, Scott, and Fowler, Plant Biotechnology, second edition, Oxford University Press, 2008. It involves contacting living plant tissue (e.g., leaf or flower tissue) with an Agrobacterium cell suspension containing a Ti-plasmid and a binary vector containing T-DNA. The entry of Agrobacterium cells into the plant tissue can be facilitated by pressure differential (e.g., using a needleless syringe) or aspiration (e.g., vacuum permeation). Alternatively, plant tissues can be sprayed with a suspension containing Agrobacterium cells and optional abrasives and surfactants, as described in, for example, WO2012019660.

[0256] For Agrobacterium-mediated transfection, the first nucleic acid molecule of the present invention can be a plasmid (e.g., a binary vector) containing a first DNA construct encoding the protein of the present invention in its T-DNA. The second nucleic acid molecule of the present invention can be a plasmid (e.g., a binary vector) containing the donor nucleic acid of the present invention in its T-DNA. The third nucleic acid molecule of the present invention can be a plasmid (e.g., a binary vector) containing a DNA construct encoding one or more gRNAs of the present invention in its T-DNA. However, binary vectors containing more than one of the said nucleic acids (e.g., the first and second nucleic acids) in a single molecule in their T-DNA can be generated. Optionally, such plasmids may additionally contain a third nucleic acid encoding one or more gRNAs in their T-DNA. If two or more nucleic acid molecules are used, each type of nucleic acid molecule can be introduced into Agrobacterium and cultured separately as a binary vector. For transformation or transfection, one, two or more Agrobacterium cultures (each containing one binary vector) can be mixed, and the mixture can be used to transform plant cells or plant cells.

[0257] Agrobacterium species can belong to *Agrobacterium tumefaciens* or *Agrobacterium rhizogenes*, which are commonly used for plant transformation and transfection and are known to those skilled in the art from common knowledge. The *Agrobacterium* strain used in the methods of this invention may contain nucleic acid molecules (Ti plasmids or binary vectors) that are the first, second, or third nucleic acid molecules, or may contain nucleic acid molecules containing two or more of the nucleic acids of this invention. One or more DNA constructs are typically present in the T-DNA of the plasmid or binary vector for introducing the nucleic acid constructs into plant cells via the secretory system of *Agrobacterium*. One or more nucleic acid constructs have T-DNA boundary sequences on at least one or both sides for transfecting the one or more plants and introducing them into plant cells or plant cells. Preferably, the one or more DNA constructs are present in the T-DNA and have T-DNA boundary sequences on both sides. In this document, the term "DNA construct" refers to a recombinant construct that contains or encodes one or more components of this invention.

[0258] The DNA construct may be present in the T-DNA of the Agrobacterium strain's Ti plasmid or binary vector. The Ti plasmid or binary vector may contain a selectable marker in addition to the T-DNA to allow cloning and genetic engineering in the bacteria. However, the T-DNA transferred to plant cells may not contain a selectable marker, if present, that would allow selection of plants or plant cells containing the T-DNA. In this embodiment, examples of selectable marker genes that should not be present in the T-DNA of the Ti plasmid or binary vector are antibiotic resistance genes or herbicide resistance genes. The method of the present invention preferably utilizes transient transfection. In this embodiment, the method of the present invention does not include the step of selecting plant cells or plants incorporating one or more nucleic acid molecules of the present invention by using such antibiotic resistance genes or herbicide resistance genes. Therefore, in this embodiment, it is not necessary to incorporate antibiotic resistance genes or herbicide resistance genes into plant cells or plants. However, suitable markers can be used to select or identify editing events and cells in which editing has occurred.

[0259] Agrobacterium-mediated gene transfer and the vectors used therein are known to those skilled in the art, for example from the references cited herein or from textbooks on plant biotechnology such as Slater, Scott, and Fowler, Plant Biotechnology, second edition, Oxford University Press, 2008. The Agrobacterium strains that can be used in this invention are those commonly used in the art for transfecting or transforming plants. Typically, on the one hand, binary vector systems and binary strains are used, i.e., the vir gene required to transfer T-DNA into plant cells is used, and on the other hand, the T-DNA is on a separate plasmid. Examples of available Agrobacterium strains are given in the article on binary Agrobacterium strains and vector systems, Hellens et al., Trends in Plant Science 5 (2000) 446-451. In the case of binary Agrobacterium strains, the plasmid containing the vir gene is referred to as a “vir plasmid” or “vir helper plasmid.” The plasmid containing the T-DNA to be transfected is a so-called binary vector, which can be the “DNA molecule” or “vector” of this invention.

[0260] Therefore, the present invention also provides Agrobacterium cells containing the first nucleic acid molecule of the present invention. Notably, the present invention provides Agrobacterium cells comprising a plasmid containing a first DNA construct encoding a polynucleotide in its T-DNA.

[0261] Co-transfection via Agrobacterium can be achieved by preparing two or more different Agrobacterium cultures, the first containing a first nucleic acid molecule (Ti plasmid or binary vector), construct, or vector encoding a fusion protein, and the second containing a second nucleic acid molecule. A third Agrobacterium culture containing a third nucleic acid molecule can also be prepared. Suspensions of these Agrobacterium cultures can be grown separately and mixed before transfection. The Agrobacterium suspension can be produced as follows. Nucleic acid molecules or vectors can be transformed into Agrobacterium strains, and the transformed Agrobacterium cultures can preferably be grown under selective pressure to maintain the relevant nucleic acid molecules. In one method, the Agrobacterium strain to be used in the method of the present invention is inoculated into a culture medium and grown to a high cell concentration. Agrobacterium is typically grown to a cell concentration corresponding to an OD of at least 1 at 600 nm, typically about 1.5. This highly concentrated Agrobacterium suspension is then diluted to achieve the desired cell concentration. To dilute the highly concentrated Agrobacterium suspension, water or Agrobacterium infiltration medium can be used. The water may contain a buffer or salt. The water may further contain surfactants or wetting agents. Optionally, the concentrated Agrobacterium suspension can be diluted with water, and any additives such as surfactants and optional buffers may be added after or during the dilution process. The separately produced suspensions for co-transfection can then be mixed, and the mixed suspension can be used to transfect plant cells or plant cells.

[0262] To transfect plant cells in a cell culture, an Agrobacterium suspension can be added to the plant cell culture. To transfect selected parts of a plant, such as leaves, the commonly known Agrobacterium-mediated agroinfiltration method can be used, whereby a pressure differential inserts the Agrobacterium suspension into the plant tissue. For example, a needleless syringe containing the Agrobacterium suspension can be used to pressurize the suspension into the plant tissue. In another Agrobacterium-mediated agroinfiltration method, the entire plant or a major part of the plant is inverted and immersed in the Agrobacterium suspension, a vacuum is applied, and then it is rapidly released, thereby inserting the Agrobacterium suspension into the plant tissue.

[0263] Kits for editing endogenous DNA

[0264] This invention also includes a kit for editing endogenous DNA at target sites in eukaryotic cells. The kit comprises at least two parts: a donor nucleic acid, donor construct, or second nucleic acid molecule as described in the “Donor Nucleic Acids” section, and a protein of the invention as described in the “Proteins and Fusion Proteins of the Invention” section. The kit contains the protein of the invention, either in the form of an expressed protein or as a nucleic acid molecule containing a polynucleotide encoding said protein (e.g., a fusion protein). The kit may further comprise eukaryotic cells. When the donor nucleic acid and protein contained in the kit of the invention are provided to the same eukaryotic cell, the kit can edit the endogenous DNA of the eukaryotic cell as described herein.

[0265] If desired, the kit may also contain one or more gRNAs or one or more nucleic acid molecules encoding said gRNAs, wherein said gRNAs bind to the site-specific endonuclease portion of said protein.

[0266] The present invention also provides a kit comprising a nucleic acid molecule containing a polynucleotide encoding the first protein subunit and a nucleic acid molecule containing a polynucleotide encoding the second protein subunit.

[0267] Furthermore, the present invention also provides a kit for editing endogenous DNA at target sites in eukaryotic cells or eukaryotic organisms, wherein the kit comprises a fusion protein containing the UL12 exonuclease and a donor nucleic acid. In this specific embodiment, the kit comprises:

[0268] - The donor nucleic acid of the present invention or the donor construct of the present invention comprising said donor nucleic acid, and

[0269] - A fusion protein used for editing endogenous DNA, which contains site-specific endonucleases and 5'-3' exonucleases.

[0270] The site-specific endonuclease and the 5'-3' exonuclease are fused via a polypeptide linker, and

[0271] The length of the polypeptide linker is 25 amino acids or more, preferably 30 amino acids or more, more preferably 40 amino acids or more, even more preferably 50 amino acids or more, and most preferably 60 amino acids or more.

[0272] The site-specific endonucleases mentioned above are preferably as defined above.

[0273] The 5'-3' exonuclease is a protein with the following amino acid sequence or containing the following amino acid sequence:

[0274] (i) The amino acid sequence defined in SEQ ID NO: 32 (UL12-1) or SEQ ID NO: 33 (UL12-2), or

[0275] (ii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence identity with the amino acid sequence defined in SEQ ID NO: 32 or SEQ ID NO: 33, or

[0276] (iii) An amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence defined in SEQ ID NO: 32 or SEQ ID NO: 33, or

[0277] (iv) An amino acid sequence with 1 to 120 amino acid substitutions, additions, insertions and / or deletions, more preferably 1 to 93 amino acids, even more preferably 1 to 62 amino acids, and even more preferably 1 to 46 amino acids, and most preferably 1 to 31 amino acid substitutions, additions, insertions and / or deletions, compared to the amino acid sequence defined in SEQ ID NO: 32 or SEQ ID NO: 33.

[0278] Methods for modifying endogenous DNA in eukaryotic cells

[0279] The present invention also includes methods for editing said endogenous DNA at target sites. One method involves inserting a target nucleotide sequence into the endogenous DNA of a eukaryotic cell at a target site, and / or deleting a nucleotide sequence segment at a target site. Another method involves modifying the endogenous DNA of a eukaryotic cell at a target site. All of these methods involve providing the donor nucleic acid and protein of the present invention to a eukaryotic cell or organism. An endonuclease of said protein (e.g., a fusion protein) then cleaves the DNA at the target site, and an exonuclease of said protein treats the cleaved DNA to produce a 3' overhang, which can invade the homologous arm of the donor nucleic acid to induce DNA modification via homology-mediated repair (HDR). The DNA modification can result in the insertion of an insertion sequence carried within the donor nucleic acid into the endogenous DNA. Thus, depending on the design of the donor nucleic acid, the methods of the present invention allow for the insertion, substitution, and deletion of one or more base pairs in the endogenous DNA of a eukaryotic host cell. The principles and methods of the protein and donor nucleic acid of the present invention, as well as the insertion of the insertion sequence for inserting a repair template into endogenous DNA, are described in the preceding sections.

[0280] Methods of inserting long nucleotide fragments into the genome

[0281] The present invention also provides a method for inserting a nucleotide fragment of at least 5,000 base pairs into the genome of a eukaryotic cell at a target site. The method includes providing the protein of the present invention and a donor nucleic acid to a eukaryotic cell, wherein the donor nucleic acid contains an insert sequence of at least 5,000 nucleotides in length, and wherein the eukaryotic cell should have a functional HDR pathway. In one embodiment, the donor nucleic acid contains an insert sequence of 7,000 nucleotides, and the 7,000 nucleotides of the insert sequence can be inserted into the endogenous DNA of the eukaryotic cell at the target site. In another embodiment, the insert sequence is 15,000 nucleotides in length, and the 15,000 nucleotides of the insert sequence are inserted into the endogenous DNA at the target site. In a further embodiment, the insert sequence is 20,000 nucleotides in length, and the 20,000 nucleotides of the insert sequence are inserted into the endogenous DNA at the target site. There is no upper limit to the insert length; however, the frequency of successful insertion events may decrease with increasing insert sequence length.

[0282] This invention also provides a method for modifying endogenous DNA of eukaryotic cells or eukaryotic organisms at a target site, independent of HDR and donor nucleic acid / donor sequence. This method is particularly suitable for inducing random deletions of one or more consecutive nucleotides in endogenous DNA at a target site, such as in a non-coding region or a cis-active element. In one embodiment, the method includes providing the cell or organism with the following:

[0283] The protein of the present invention, or the nucleic acid molecule of the present invention, or the nucleic acid construct, plasmid, or vector of the present invention, or the kit of the present invention. As a result, the method produces modifications to endogenous DNA, wherein the modifications include the random deletion of two or more nucleotide sequences in the endogenous DNA upstream and / or downstream of the target site. Example

[0284] Example 1: Used for translating GUS and Benzoic nicotinic glycerol kinase NbPGK Fusion donor construct design of (Niben101Scf05688g08010.1)

[0285] First, we identified putative single guide RNA (sgRNA) target sites in the PAM-in direction near the stop codon in the NbPGK genome sequence (via CRISPOR (http: / / crispor.tefor.net / ) and CRISPR-P v2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ); Figure 1To construct the donor construct, we amplified and cloned approximately 1 kb upstream and downstream of the stop codon as 5' and 3' homologous arms (HAs), respectively. Mutations were made in the sgRNA target sequence within the donor fragment to prevent Cas9-mediated donor cleavage (before and / or after integration). The amplified fragment was adapted for Golden-Gate (GG) cloning by removing the internal BsaI or BpiI sites using appropriate oligonucleotides. In the donor (5' HA), the stop codon of NbPGK was deleted to allow translational read-trough, resulting in a translated NbPGK-GUS fusion. Orthogonal target sites of sgRNA_Csy4-3 were integrated upstream and downstream of the donor fragment in a PAM-out orientation for cleavage, processing, and release of the donor fragment. The donor fragment was assembled via GG-cloning and delivered as T-DNA in *Agrobacterium tumefaciens* (GV3101 pMP90 strain)-mediated transient expression in *N. benthamiana* (Nb) leaves.

[0286] The polynucleotide sequence of the donor fragment (PGK-GUS) is the polynucleotide sequence of SEQ ID NO: 1:

[0287]

[0288]

[0289]

[0290]

[0291] In the polynucleotide SEQ ID NO: 1 shown above, the format has the following meaning:

[0292] Left border of T-DNA; Target sequences used for Cas9 cleavage outside the donor fragment; 5'HA+3'HA: 5' and 3' homologous arms; sgR-PGK1+2 mutant PAM ): The sequence of the PGK locus that is targeted by a single guide RNA but mutated in the donor; (Intron): GUS gene containing introns; The right border of T-DNA

[0293] Example 2: Design of SpCas9 exonuclease fusion complex and sgRNA

[0294] The exonuclease was codon-optimized for Nicotiana benthamiana and synthesized as a Level-1 GG-module with a matching dangling splint. Figure 2The LF2 adapter fragment was amplified by primer extension and then assembled into Level 1 modules, producing 2xLF2 or 4xLF2 (see sequences below). A 432bp fragment from the XTEN adapter (144aa) was synthesized as a Level 1 GG-module. Using the 432bp XTEN fragment as a template, shorter variants of the XTEN adapter (XTEN16 and XTEN40) were generated via PCR and assembled into Level 1 GG modules. The exonuclease and adapter modules were then assembled together using GG cloning to form Level 0 modules. Figure 2 The obtained exonuclease-adaptor level 0 module was assembled into a level 1 unit along with 2×35S (short), ω translation enhancer, SpCas9i, and tOCS terminator as transcription units. The level 1 expression vector was transformed into Agrobacterium rhizogenes (GV3101 pMP90) and used for transient expression in tobacco leaves.

[0295] N-terminal exonuclease-Cas9 fusion

[0296] The general structure of the construct encoding the target protein is as follows ( Figure 2 A):

[0297] RB (Right Boundary of T-DNA) -2x35S short (dicecauliflower mosaic virus 35S promoter, short form)-translation enhancer (ω-translation enhancer from tobacco mosaic virus) -Exonuclease-Connector -SpCas9 (SpCas9 form with introns) / LbCas12a (LbCas12a form with introns) -tOCS (OCS transcription terminator) -LB (left border of T-DNA).

[0298] sequence - indicates a cantilever used in Golden Gate cloning to assemble different modules.

[0299] The polynucleotide sequence of the promoter-enhancer module GGAG_2x35Ss_TACT-ΩAATG is shown in SEQ ID NO: 2 below:

[0300]

[0301]

[0302] The polynucleotide sequence of the Cas9 module AGGT_NLS-SpCas9i-NLS*_GCTT used for N-terminal fusion is shown in SEQ ID NO: 3 below (* indicates a stop codon, NLS indicates a nuclear localization signal, bold letters correspond to coding sequences, and normal letters correspond to introns):

[0303]

[0304]

[0305]

[0306]

[0307]

[0308] The polynucleotide sequence of the Cas12a module AGGT_NLS-LbCas12ai-NLS*_GCTT used for the N-terminal fusion complex is shown in SEQ ID NO: 57 below (* indicates a stop codon, NLS indicates a nuclear localization signal, bold letters correspond to coding sequences, and normal letters correspond to introns):

[0309]

[0310]

[0311]

[0312]

[0313]

[0314] Transcription Termination Submodule GCTT_ tOCS The polynucleotide sequence of _CGCT is shown in SEQ ID NO: 17 below:

[0315]

[0316] In the polynucleotides shown above with SEQ ID NO: 2, 3 and 17, the format has the following meaning:

[0317] 2×35S short: 35S promoter of cauliflower mosaic virus, short form. Translation enhancer : The ω-translation enhancer from tobacco mosaic virus, NLS-SpCas9 (intron)-NLS: The SpCas9 form with introns and N-terminal and C-terminal nuclear localization sequences (NLS), tOCS OCS transcription terminator Golden Gate clonal overhang.

[0318] In the assembled Level 1 construct, the sequences of SEQ ID NO: 2, 3, and 17 are associated with what is known as the "Exo-connector". Figure 2 A contains a combination of exonucleases and polypeptide linkers. The exo-linker is cloned from GG (Golden Gate) clones. Figure 2 The first-level segments “Exo” and “connector” shown in Figure A are assembled.

[0319] The polynucleotide sequences of exonucleases and various linkers are as follows:

[0320] SEQ ID NO:4: Polynucleotide encoding the linker TTCG_2xLF2_AGGT

[0321]

[0322] SEQ ID NO:5: Polynucleotide encoding the linker TTCG_4xLF2_AGGT

[0323]

[0324] SEQ ID NO:6: Polynucleotide encoding the linker TTCG_XTEN144_AGGT

[0325]

[0326] SEQ ID NO:7: Polynucleotide encoding the linker TTCG_XTEN40_AGGT

[0327]

[0328] SEQ ID NO:8: Polynucleotide encoding the linker TTCG_XTEN16_AGGT

[0329] SEQ ID NO:9: Polynucleotide encoding T7 exonuclease (AATG_T7_TTCG) for N-terminal exonuclease-Cas9 fusion.

[0330]

[0331] SEQ ID NO:10: Polynucleotide encoding T5 exonuclease (AATG_T5_TTCG) for N-terminal exonuclease-Cas9 fusion.

[0332]

[0333]

[0334] SEQ ID NO:11: Polynucleotide encoding UL12 exonuclease (AATG_UL12_TTCG) for N-terminal exonuclease-Cas9 fusion.

[0335]

[0336]

[0337]

[0338] SEQ ID NO:12: Polynucleotide encoding UL12-2 exonuclease (AATG_UL12-2_TTCG) for N-terminal exonuclease-Cas9 fusion.

[0339]

[0340]

[0341] SEQ ID NO:13: Polynucleotide encoding the Epstein-Barr virus BGLF5 exonuclease (AATG_BGLF5_TTCG) for the N-terminal exonuclease-Cas9 fusion.

[0342]

[0343]

[0344] SEQ ID NO:14: Polynucleotide encoding the varicella-zoster virus (Dumas strain) exonuclease (DUMAS) for the N-terminal exonuclease-Cas9 fusion (AATG_DUMAS_TTCG)

[0345]

[0346]

[0347] SEQ ID NO:15: Polynucleotide encoding the Marek's disease MD5 exonuclease (AATG_MD5_TTCG) for the N-terminal exonuclease-Cas9 fusion.

[0348]

[0349]

[0350]

[0351] SEQ ID NO:58: Polynucleotide encoding Shutoff basic exonuclease SOX (AATG_SOX_TTCG) for N-terminal exonuclease-Cas9 fusion.

[0352]

[0353]

[0354] SEQ ID NO:59: Polynucleotide encoding equine herpesvirus 1 (Ab4p strain) exonuclease (AATG_AB4P_TTCG) for N-terminal exonuclease-Cas9 fusion.

[0355]

[0356]

[0357] SEQ ID NO:60: Polynucleotide encoding the Papiineα herpesvirus 2 (OU1-76 strain) exonuclease AATG_PapE_TTCG exonuclease for the N-terminal exonuclease-Cas9 fusion.

[0358]

[0359] SEQ ID NO:61: Polynucleotide encoding the Lyri fox-associated alpha herpesvirus exonuclease (PiE) AATG_PiE_TTCG for the N-terminal exonuclease-Cas9 fusion.

[0360]

[0361]

[0362]

[0363] Table 1: Primary assembly of modules for N-terminal labeled SpCas9 (exonuclease-linker-SpCas9).

[0364]

[0365]

[0366] First, any given exonuclease-adaptor combination is assembled from level 1 to level 0. The corresponding exonuclease-adaptor fusion (level 0) is combined with other given level 0 modules (2x35Ss_Ω enhancer_exonuclease-adaptor_NLS-SpCas9i-NLS_tOCS) and assembled into a GG-compatible MoClo level 1 T-DNA vector for Agrobacterium-mediated transient expression in plants (including LB and RB sequences for T-DNA delivery).

[0367] C-terminal Cas9-exonuclease fusion

[0368] The general structure of the construct encoding the protein of this invention is as follows ( Figure 2 B):

[0369]

[0370] The polynucleotide sequence of the Cas9 module AATG_NLS-SpCas9i-NLS_TTCG used for the C-terminal fusion complex is shown in SEQ ID NO:16 below:

[0371]

[0372]

[0373]

[0374]

[0375]

[0376] In the polynucleotide sequence of SEQ ID NO:16, the format has the following meaning:

[0377] NLS-SpCas9((intron)-NLS

[0378] The polynucleotide sequence of the LbCas12a(D156R) module AATG_NLS-LbCas12ai-NLS_TTCG for the C-terminal fusion complex is shown in the following SEQ ID NO:62:

[0379]

[0380]

[0381]

[0382]

[0383] In the assembled Level 1 construct, the sequences of SEQ ID NO: 2, 16, and 17 are consistent with... Figure 2 The "linker-Exo" in B is a combination of fragments containing a polypeptide linker and an exonuclease. Linker-Exo is cloned from GG (Golden Gate) clones... Figure 2 The first-level segments "connector" and "Exo" shown in B are assembled.

[0384] Adapters for C-terminal exonuclease-Cas9 fusion proteins (underlined box) )

[0385] SEQ ID NO:18: Polynucleotide encoding the linker TTCG_2xLF2_AATG

[0386]

[0387] SEQ ID NO:19: Polynucleotide encoding the linker TTCG_4xLF2_AATG

[0388]

[0389] SEQ ID NO:20: Polynucleotide encoding the linker TTCG_XTEN144_AATG

[0390]

[0391] SEQ ID NO:21: Polynucleotide encoding the linker TTCG_XTEN40_AATG

[0392]

[0393] SEQ ID NO:22: Polynucleotide encoding the linker TTCG_XTEN16_AATG

[0394] Exonuclease for C-terminal exonuclease-Cas9 fusion

[0395] SEQ ID NO:23: Polynucleotide encoding the T7 exonuclease (AATG_T7*_GCTT) for the C-terminal exonuclease-Cas9 fusion (* indicates a stop codon)

[0396]

[0397] SEQ ID NO:24: Polynucleotide encoding the T5 exonuclease (AATG_T5*_GCTT) for the C-terminal exonuclease-Cas9 fusion (* indicates a stop codon)

[0398]

[0399]

[0400] SEQ ID NO:25: Polynucleotide encoding UL12 exonuclease (AATG_UL12*_GCTT) for C-terminal exonuclease-Cas9 fusion (* indicates stop codon)

[0401]

[0402]

[0403] SEQ ID NO:26: Polynucleotide encoding UL12-2 exonuclease (AATG_UL12-2*_GCTT) for C-terminal exonuclease-Cas9 fusion (* indicates stop codon)

[0404]

[0405]

[0406] SEQ ID NO:63: A polynucleotide encoding the Stenotrophomonas phage IME15 exonuclease (AATG_ME15*_GCTT) for the C-terminal exonuclease-Cas9 fusion (* indicates a stop codon).

[0407]

[0408]

[0409] SEQ ID NO:64: A polynucleotide encoding the Yersinia phage phiYeO3-12 exonuclease (AATG_O3-12*_GCTT) for the C-terminal exonuclease-Cas9 fusion (* indicates a stop codon).

[0410]

[0411] SEQ ID NO:65: A polynucleotide encoding the Spirochaeta bacterium exonuclease (AATG_SpiPh*_GCTT) for the C-terminal exonuclease-Cas9 fusion complex (* indicates a stop codon).

[0412]

[0413] SEQ ID NO:66: Polynucleotide encoding Pasteurella phage vB_PmuP_PHB02 exonuclease (AATG_PhBO2*_GCTT) for C-terminal exonuclease-Cas9 fusion (* indicates stop codon)

[0414]

[0415]

[0416] SEQ ID NO:67: A polynucleotide encoding the Ralstonia phage phiITL-1 exonuclease (AATG_RaTL1*_GCTT) for the C-terminal exonuclease-Cas9 fusion (* indicates a stop codon).

[0417]

[0418]

[0419] Table 2: Level 1 Assembly of Modules for SpCas9 (SpCas9-Connector-Exo) with C-End Marking

[0420]

[0421]

[0422] First, any given adapter-exonuclease combination is assembled from level 1 to level 0. The corresponding adapter-exonuclease fusion (level 0) is combined with other given level 0 modules (2x35Ss_Ω enhancer; NLS-SpCas9i-NLS; adapter-exo; tOCS) and assembled into a GG-compatible MoClo level 1 T-DNA vector for Agrobacterium-mediated transient expression in plants (including LB and RB sequences for T-DNA delivery).

[0423] sgRNA design

[0424] Cas9 sgRNA was amplified via PCR as a Golden-Gate (GG) module. The forward primer bound the sgRNA scaffold and introduced a novel spacer sequence and a BsaI restriction site including the corresponding overhang (ATTG, where the bolded guanine is the SlU6 transcription start site). An sgRNA flip extension scaffold (Chen et al., 2013) and a subsequent 67bp U-26 terminator (Castel et al., 2018) were used as templates (pAGT6182). The reverse primer bound to the terminator sequence and contained a BsaI site including the corresponding overhang (CGCT). Figure 3 The obtained sgRNA terminator PCR fragment was combined with the U6 promoter (SlU6, pAGT5824) from tomato (Solanum lycopersicum) into a primary T-DNA vector via GG-cloning. The primary expression vector was transformed into Agrobacterium tumefaciens (GV3101 pMP90) and used for transient expression in tobacco leaves.

[0425] SEQ ID NO: 27: sgRNA transcription unit

[0426]

[0427]

[0428] In the polynucleotide SEQ ID NO: 27 shown above, the format has the following meaning:

[0429] SlU6: The U6 promoter from Tomato. sgRNA scaffold (SpCas9), AtU6-26-t67 The U6 promoter is from Arabidopsis thaliana.

[0430] SEQ ID NO: 28: Spacer of sgR-PGK1 (as shown in SEQ ID NO: 27):

[0431] AGCCACATATCCACTGGTGG

[0432] SEQ ID NO: 29: Spacer of sgR-PGK2 (optional spacer, which may replace spacer SEQ ID NO: 28 in SEQ ID NO: 27):

[0433] CCACTGATTATGCTGATCAC

[0434] Example 3: The fusion of GUS and NbPGK, expressed transiently in tobacco leaves via gene-targeted translation, was achieved.

[0435] To verify the efficiency of the generated SpCas9-exonuclease variant, we expressed all components (SpCas9, sgRNA, and donor fragment) in *Nicotiana benthamiana* (Nb) via Agrobacterium-mediated transient expression. Successful gene targeting should result in measurable GUS activity.

[0436] The corresponding Agrobacterium strains were grown on plates (LB agar with the corresponding antibiotics) at 28°C for 2 days. The grown bacteria were resuspended in AIM (Agrobacterium osmosis medium) until the optical density (OD) of the Cas9 variant and sgRNA / donor was measured. 600 The concentrations were 0.1 and 0.2, respectively. Equal volumes of Agrobacterium strain dilutions were mixed together (1:1:1:1, Cas9-constructor: sgR-PGK1: sgR-PGK2: donor). The Agrobacterium suspension was inoculated into Nb leaves using a needleless syringe. Four days post-inoculation, leaf samples were collected, and GUS activity was analyzed quantitatively (two leaf discs, 0.9 cm) and qualitatively (one leaf disc, 0.9 cm) according to Kay et al., 2007. A GUS construct driven by the constitutive promoter of actin 2 was inoculated separately and used as a positive control. The GUS activity produced by different Cas9-exonuclease fusions was relative to Act2p-GUS activity (…). Figure 4A Compared to Cas9, the fusions of Cas9-4LF2-UL12, UL12-2-4LF2-Cas9, and T7-4LF2-Cas9 resulted in the highest increase in GUS activity. Figure 4A Low GUS activity was detected when dCas9 was used, which was comparable to the GUS activity (background activity) of the control without any nuclease. This was determined using VHX microscopy (Keyence). Figure 4B Photographs of the stained leaf discs were taken. The leaf disc and quantitative GUS data were from the same experiment, which was repeated twice, and the results were similar.

[0437] Example 4: Analysis of exonuclease activity by in vitro processing of blunt-terminated hairpin oligonucleotides

[0438] To determine the activity of T5 and T7 phage exonucleases on blunt-terminated dsDNA, we used hairpin oligonucleotides with a linked fluorophore (Oregon Green) as described in (Nikiforov, 2014), except that the 5' end was phosphorylated. The fluorophore was quenched by stacking GC base pairs at the blunt-terminal site. Figure 5 Exonucleases process blunt ends to release fluorophores, preventing quenching, and the fluorescence can be measured (excitation at 495 nm, emission at 520 nm).

[0439] Reaction (in 100 μL):

[0440] 10 μL buffer NEB4

[0441] 2.5 μL exonuclease (1:5 dilution; 5 U)

[0442] x μL oligonucleotides (adjust to the desired final concentration (5 to 20 μM))

[0443] 2 μL DTT (250 mM)

[0444] yμL H2O to 100μL

[0445] T5 and T7 exonucleases were purchased from New England Biolabs (catalog numbers M0363 and M0263, respectively). The oligonucleotide has the nucleotide sequence: GGAAGGGCCCGCTGACAGTTTTTCTGTCAGCGGGCCCTTCC (SEQ ID NO: 37). It is phosphorylated at the 5' end and... Figure 5 The indicated position carries Oregon Green fluorescent dye.

[0446] All components were mixed in a 96-well plate on ice to prevent reaction initiation. The reaction was initiated while the plate was placed in a TECAN Spark Fusion reader. Measurements were taken at 27°C for 30 minutes.

[0447] To calculate the kinetic parameters of the T7 exonuclease, four different concentrations of hairpin oligonucleotides (5, 10, 15, and 20 μM) were used. The fluorescence values ​​of the enzyme-free control were subtracted, and the initial rate of reaction (V0) was calculated by measuring the slope of the fluorescence increase, which was linear during the initial 300 to 400 seconds (see [link to relevant documentation]). Figure 5 Then, using the Lineweaver-Burk linear representation of the Michaelis-Menten equations, K is calculated with this set of V0 values. m and K cat The values ​​are significant. It should be mentioned that the kinetic parameters calculated in this way are specific and relevant to this assay because exonucleases are processing enzymes that cleave nucleotides one after another.

[0448] Comparison of T5 and T7 ( Figure 5 This clearly demonstrates that T7 exonuclease degrades blunt-terminal oligonucleotides much faster than T5 exonuclease.

[0449] Example 5: Design of C-terminal Cas12a(D156R) exonuclease fusion variant

[0450] The construct was designed similarly to that described in Example 2. The general structure of the assembled construct is: T-DNA right boundary RB, 2×35S short, translation enhancer, NLS-LbCas12a(D156R)-NLS (intron), adapter-exonuclease, tOCS, T-DNA left boundary LB.

[0451] The polynucleotide sequence of the Cas12a module AATG_NLS-LbCas12a(D156R)-NLS(intron)_TTCG for the C-terminal fusion is shown in the following SEQ ID NO: 38:

[0452]

[0453]

[0454]

[0455]

[0456]

[0457] In the polynucleotide shown above, SEQ ID NO: 38, the format has the following meaning: NLS-LbCas12a(D156R)-NLS,INTRON, Table 3: Level 1 Assembly of Modules Used for C-End Marking LbCas12a (LbCas12a(D156R)i-Connector-Exo)

[0458]

[0459]

[0460] * indicates a stop codon (as mentioned above).

[0461] First, any given adapter-exonuclease combination is assembled from Level 1 to Level 0. The corresponding adapter-exonuclease fusion (Level 0) is then combined with other Level 0 modules (2x35Ss_Ω enhancer_NLS-LbCas12a(D156R)i-NLS_adaptor-exo_tOCS) and assembled into a GG-compatible MoClo Level 1 T-DNA vector for Agrobacterium-mediated transient expression in plants (including LB and RB sequences for T-DNA delivery).

[0462] Example 6: Design of N-terminal Cas12a(D156R) exonuclease fusion variant

[0463] The construct was designed similarly to that described in Example 2. The exonuclease and adapter can be as described in Example 2. The general structure of the construct to be assembled is: T-DNA right border-RB, 2×35S short, translation enhancer, exonuclease-adaptor, NLS-LbCas12a(D156R)-NLS (intron), tOCS, T-DNA left border LB.

[0464] The polynucleotide sequence of the Cas12a module AGGT_NLS-Cas12a(D156R)-NLS(intron)_GCTT for the N-terminal fusion complex is shown in the following SEQ ID NO: 39:

[0465]

[0466]

[0467]

[0468]

[0469]

[0470] In the polynucleotide SEQ ID NO: 39 shown above, the format has the following meaning: NLS-LbCas12a(D156R)-NLS (intron)

[0471] Table 4: Level 1 Assembly of Modules for LbCas12a (Exo-connector-LbCas12a) with N-terminal Marking

[0472]

[0473]

[0474] First, any given exonuclease-adaptor combination is assembled from level 1 to level 0. The corresponding exonuclease-adaptor fusion (level 0) is combined with other given level 0 modules (2x35Ss_Ω enhancer_exonuclease-adaptor_NLS-SpCas9i-NLS_tOCS) and assembled into a GG-compatible MoClo level 1 T-DNA vector for Agrobacterium-mediated transient expression in plants (including LB and RB sequences for T-DNA delivery).

[0475] Example 7: Transgenic Tobacco Benzoinus mosaic virus (TMV) using GFP ) Reported strains

[0476] In addition to the aforementioned GUS-based reporting system, a GFP-based virus reporting system has also emerged. Figure 6A schematic diagram of a TMV-based HDR reporter system using GFP is shown. The genome of *Nicotiana benthamiana* is transgenic and contains an insert cassette containing a TMV-based HDR reporter system using GFP. This reporter system produces GFP fluorescence when the donor sequence from the donor nucleic acid (the donor) is successfully and correctly integrated into the plant genome. The genome of the TMV present in the insert cassette is modified: (i) the sequence encoding the capsid protein (CP) is exchanged for a sequence encoding GFP, and (ii) the replicase RdRP contains a 3.8 kb deletion substituted by a 76 bp attB site. MP is intact and facilitates viral cell-to-cell transmission (signal transduction), which allows for the visual observation of GFP expression derived from single-cell HDR events (one GFP spot equals one single-cell HDR event). RdRP is essential for viral replication and the production of secondary transcripts (MP and GFP) from the subgenomic promoter. The exchange of CP for GFP prevents the viral genome from being packaged into viral particles (non-infectious virus) and allows for high RdRP-dependent GFP expression rates. Viral replication (GFP expression) occurs only when the damaged RdRP is repaired via precise HDR insertion of the provided donor DNA. Four Cas9 sgRNA targets, specific to the attB site, are used to induce DNA double-strand breaks (DSBs) in either the PAM-in (a combination of PAM-In1 and PAM-In2 sgRNAs) or PAM-out (a combination of PAM-out1 and PAM-out2 sgRNAs) direction. DSB repair via homology-mediated repair (HDR) using the provided donor nucleic acid leads to the remodeling of the TMV replicase (RdRP) and subsequent GFP production (as HDR readout). See details... Figure 6 In the following embodiments, the reporting system is used to measure HDR frequencies.

[0477] Example 8: Cas9 fusion with exonuclease leads to increased HDR in plants.

[0478] Report using transgenic Nicotiana benthamiana TMV from Example 7 strainHDR efficiency was analyzed. As described in Example 3, T-DNA carrying transcription units expressing (i) the Cas9 exonuclease fusion protein, (ii) sgRNA, and (iii) donor DNA was transiently delivered to the leaves of *Nicotiana benthamiana* plants via Agrobacterium-mediated transient transformation using a needleless syringe. GFP fluorescence was monitored under UV light generated by a handheld lamp (Blak-Ray B100A from UVP) at 3 or 6 days post-inoculation (dpi), and photographs were taken using a digital camera (Canon EOS 700D). The Cas9 fused with the exonuclease was compared with WT Cas9 (denoted as "Cas9") and inactivated Cas9 (denoted as "dCas9") as controls. The exonuclease was fused to the C-terminus of Cas9 using a 4LF2 adapter (Cas9-4LF2-X). Figure 7 In the study, the fusion of UL12 and T7 with Cas9 resulted in the strongest HDR enhancement. Shortly after 3 dpi, a high rate of HDR events was observed with Cas9 fused with UL12. After 6 dpi, GFP fluorescence saturated in tissues expressing Cas9 fused with UL12 and T7. Cas9 expression at 6 dpi resulted in only a few HDR events. See also Figure 7 .

[0479] Example 9: Quantifying HDR events using GFP point counting

[0480] To quantify HDR events, as described in Example 8, images of leaves from three inoculated plants of *Nicotiana benthamiana* were captured at 3 dpi and 6 dpi using a digital camera, while the leaves were illuminated in darkness using a handheld UV lamp. All constructs were inoculated onto a single leaf in a randomized manner on each leaf. To assess equivalent GFP dot counts, GFP fluorescent dots within regions of the same size matched to each inoculation site were manually counted (inoculation site; GFP dot equals HDR event). GFP signal saturation (undefined -nd) is displayed. The number of GFP dots in the Cas9-expressing tissue (6 dpi) did not exceed the number of GFP dots in the Cas9-UL12-expressing tissue (3 dpi). See also Figure 8 .

[0481] Example 10: Genotyping of HDR events using PCR

[0482] Genomic DNA from *Tobacco Bengal* leaves was isolated at 4 days post-contraction (dpi) from tissues expressing dual PAM In sgRNA, donor nucleic acid (donor), and the corresponding Cas9 exonuclease fusion. Details are described in... Figure 9In the middle, primer pair P1 (composed of primers 1F and 1R) was used to monitor the on-target NHEJ event (small deletion). Primer pairs P2 (2F and 2R) and P3 (3F and 3R) were used to amplify the upstream and downstream of the HDR ligation, respectively. Cas9 resulted in a fragment deletion flanking the sgRNA target site (small band). The intensity of this fragment was negatively correlated with the number of GFP spots. For Cas9 variants fused to UL12, T5, and T7, ligation of the HDR event was confirmed on both sides. Sequencing confirmed accurate repair (data not shown). See details. Figure 9 .

[0483] Example 11: The exonuclease domain of Exo1 (Exo1ΔC) fused with Cas9 only slightly increases HDR efficiency.

[0484] HDR efficiency was analyzed using the transgenic Nicotiana benthamiana TMV reporter line according to Examples 7 and 8. GFP fluorescence was monitored at 5 dpi. Cas9 fused with exonuclease was compared with WT Cas9 and inactivated Cas9 (dCas9) as controls. X-4LF2-Cas9 showed that the exonuclease was fused to the N-terminus of Cas9 using the 4LF2 linker. The exonuclease domain (Exo1ΔC) of the Exo1 fused Cas9 resulted in only a slight increase in HDR events compared to Cas9. Cas9 fused with UL12 and T7 performed better than Cas9 fused with Exo1ΔC. See also Figure 10 .

[0485] Example 12: Comparison of UL12-homogeneities in HDR

[0486] exist Figure 11 The figure above shows the amino acid (aa) sequence identity of the analyzed UL12-homophores. HDR efficiency was analyzed using the transgenic Nicotiana benthamiana TMV reporter lines from Examples 7 and 8. Figure 11 Among them, UL12 homologs with 50% or higher sequence identity, such as PiE (SEQ ID NO: 44) or PapE (SEQ ID NO: 43), showed HDR-ratios that were comparable to or higher than those of UL12 (SEQ ID NO: 32) or UL12-2 (SEQ ID NO: 33). Figure 12 Provided for Figure 11 Quantitative GFP spot counting analysis was performed. GFP spot counting was conducted as described in Example 9. Figure 12 This indicates that PapE (SEQ ID NO: 43) and PiE (SEQ ID NO: 44) fused with Cas9 exhibit increased HDR efficiency compared to UL12 and UL12-2. UL12 homologs with sequence identity below 49% showed lower HDR-ratios than UL12 or UL12-2. See also Figure 11 , 12, 15 and 17. UL12=SEQ ID NO:32, UL12-2=SEQ ID NO:33, PapE=SEQ ID NO:43, PiE=SEQ ID NO:44, AB4P=SEQ ID NO:69, MD5=SEQ ID NO:36, Dumas=SEQ ID NO:35, BGLF5=SEQ ID NO:34, SOX=SEQ ID NO:68.

[0487] Example 13: Comparison of T7-homogeneities in HDR

[0488] exist Figure 13 The figure above shows the amino acid (aa) sequence identity of the analyzed T7-homophores. HDR efficiency was analyzed using the transgenic Nicotiana benthamiana TMV reporter lines of Examples 7, 8, and 12. T7 homologs ME15 (SEQ ID NO: 45) (87% sequence identity) and SpiPh (SEQ ID NO: 46) (65% sequence identity) showed increased HDR-ratios compared to T7 (SEQ ID NO: 30). T7 homolog ME15 (SEQ ID NO: 45) showed HDR efficiency comparable to UL12 (SEQ ID NO: 32) and UL12-2 (SEQ ID NO: 33), and... Figure 13 neutralization Figure 14 The GFP dot count analysis showed a higher value than T7. Figure 14 Provided for Figure 13 Quantitative GFP spot counting analysis was performed. GFP spot counting was conducted as described in Example 9. Figure 13 and 14 In this study, the T7 homolog SpiPh (SEQ ID NO: 46) exhibited higher HDR efficiency than T7. See also: Figure 16 and 17 . UL12 / UL12-1=SEQ ID NO:32, UL12-2=SEQ ID NO:33, T7=SEQ ID NO:30, ME15 / IME15=SEQ ID NO:45, O3-12 / YerO3-12=SEQ ID NO:70, SpiPh=SEQ ID NO:46, PHBO2=SEQ ID NO:71, RaTL1 / RaITL1=SEQ ID NO:72.

[0489] Example 14: Comparison of exonuclease activities of monomeric exonucleases using blunt-ended DNA substrates

[0490] See Figure 18(A) Fluorescently labeled hairpin oligomers were used to evaluate exonuclease activity against blunt-ended DNA. The fluorophore was quenched by stacking onto the GC-bonds at the flat ends. 5' to 3' cleavage by the exonuclease resulted in the release of the fluorophore and subsequent fluorescence. (B) Assays were performed according to Example 4, with labeled oligomers (10 μM) incubated with different exonucleases at 28°C for more than 30 minutes. T5 (NEB; #M0363), T7 (NEB; M0263S), and recombinant His-labeled UL12 had been purified from *E. coli*. Figure 18 The rapid increase in fluorescence indicates that UL12 and T7 possess higher exonuclease activity and a preference for blunt-terminated DNA substrates compared to T5. See also Figure 18 . UL12=SEQ ID NO:32, T7=SEQ ID NO:30, T5=SEQ ID NO:31.

[0491] Example 15: Comparison of nuclei of monomers (T5 and T7) and trimer (LaExo) exonucleases using blunt-ended DNA substrates. acid exonuclease activity

[0492] See Figure 19 (A) Fluorescently labeled hairpin oligomers were used to assess exonuclease activity against blunt-ended DNA. The fluorophore was quenched by stacking onto the GC-bonds at the flat ends. 5' to 3' cleavage by the exonuclease resulted in the release of the fluorophore and subsequent fluorescence. (B) Assays were performed according to Example 4, with the following minor changes: LaExo from NEB required its own specific buffer (LaExo buffer). T5-exo and T7-exo experiments were performed using NEB buffer 4, and LaExo experiments were performed using LaExo-specific buffer (NEB). Reactions were performed simultaneously in the same 96-well plate.

[0493] Reactions T5-Exo and T7-Exo (at 100) μ L ):

[0494] 10 μL buffer NEB4

[0495] 1 μL exonuclease (T5 or T7) (10 U)

[0496] x μL oligonucleotides (adjusted to the desired final concentration (10 μM))

[0497] 2 μL DTT (250 mM)

[0498] yμL H2O to 100μL

[0499] The reaction of LaExo (at 100) μ L ) ：

[0500] 10 μL buffer LaExo

[0501] 2 μL LaExo exonuclease (10 U)

[0502] x μL oligonucleotides (adjusted to the desired final concentration (10 μM))

[0503] 2 μL DTT (250 mM)

[0504] yμL H2O to 100μL

[0505] The labeled oligomers were incubated with different exonucleases at 28°C for more than 30 minutes. T5 (NEB; M0363), T7 (NEB; M0263S), and LaExo (NEB; M0262S). Figure 19 The rapid increase in fluorescence indicates high exonuclease activity and a preference for blunt-ended DNA substrates. T7 and LaExo (λ exonuclease) showed higher exonuclease activity for blunt-ended DNA substrates than T5 (see [link to T5]). Figure 19 However, as in Example 16 and Figure 20 As demonstrated in the study, the fusion of the trimer λ exonuclease (LaExo) with Cas9 exhibited lower HDR efficiency than the fusion of Cas9 with UL12.

[0506] Example 16: Co-expression of core-localized LaExo (referred to as N-LaExo or LaExo-N) cannot increase the fusion of Cas9. LaExo activity

[0507] HDR efficiency was analyzed using the aforementioned transgenic Nicotiana benthamiana TMV reporter line and methods. GFP fluorescence was monitored at 5 days post-inoculation (dpi). Fusion of trimeric LaExo (λ exonuclease) with Cas9 showed lower HDR efficiency than fusion of UL12 with Cas9. Co-expression of nuclear-localized LaExo (nuclear localization via nuclear localization signal (NLS); referred to as N-LaExo or La-Exo-N) did not increase HDR efficiency. See also Figure 20 N-LaExo and LaExo-N represent the nuclear localization signal (NLS) fused to the N- or C-terminus of LaExo, respectively.

[0508] Example 17: Determination of the minimum homologous arm length

[0509] HDR efficiency was analyzed using the aforementioned transgenic Nicotiana benthamiana TMV reporter line and methods. GFP fluorescence was monitored on day 5 post-inoculation (dpi). As shown, the donor nucleic acids differed in homologous arm (HA) length (1000 bp, 500 bp, 250 bp, 100 bp). The smallest homologous arm length of 250 bp indicated a slight increase in HDR efficiency. See also... Figure 21 .

[0510] Example 18: Cas12a-exonuclease fusion leads to increased HDR in plants

[0511] The HDR efficiency of the Cas12a-exonuclease fusion protein was analyzed using the aforementioned transgenic Nicotiana benthamiana TMV reporter line and methods. Fluorescence was monitored at 5 dpi. WT Cas12a and no endonuclease were used as controls. Note that Cas12a produces “staggered” cuts with overhangs on double-stranded DNA, unlike the “blunt-end” cuts produced by Cas9. See also Figure 22 See also Example 19.

[0512] Example 19: HDR efficiency of Cas12a exonuclease fusion protein estimated by GFP dot counting.

[0513] The quantitative method described in Example 9 is derived from Example 18 / Figure 22 HDR events were observed. To quantify HDR events, images of leaves from three inoculated plants of *Nicotiana benthamiana* were captured at 3 dpi. All constructs were inoculated onto a single leaf, at different locations within each plant. To assess the equivalent number of GFP fluorescent spots, counting was performed within confined regions of the same size. Unlike the exonuclease fusion with Cas9, the Cas12a fusion with T5 resulted in increased HDR compared to the Cas12a fusion with T7. The Cas12a fusion with UL12 resulted in the highest increase in HDR efficiency. See also Figure 23 dCas12a refers to deactivated Cas12a.

[0514] Example 20: Comparative analysis of NHEJ cleavage patterns of Cas9- and Cas12a- exonuclease fusion proteins

[0515] See Figure 24 By Agrobacterium-mediated expression (as described in Examples 7 and 8), the corresponding Cas9- and Cas12a-exonuclease fusion proteins and sgRNA (Cas9) / crRNA (Cas12a) (PAM-in or PAM-out) were transiently expressed in the leaves of transgenic Nicotiana benthamiana TMV-reporter lines in the presence and absence of (+D-donor DNA) and donor DNA. Genomic DNA was isolated from the plant leaf tissues expressing the corresponding nuclease-exonuclease fusion proteins and sgRNA / crRNA at 4 dpi and used as a template for mid-target amplification using primer pair P1 (primers 1F and 1R). Cas9-mediated cleavage resulted in the deletion of fragments flanking the dual sgRNA target sites ( Figure 24Smaller, additional bands; indicating NHEJ; e.g., a 44bp deletion between PAM-In1 and PAM-In2). Cas12a does not result in a significant deletion, indicating a wider deletion range or a reduction in the typically NHEJ-mediated deletion. Cas9-exonuclease fusions result in a wider deletion size, leading to a visible, blurred band rather than a distinct band. Figure 24 The reduced precision missing frequency is correlated with the increased HDR. See also Examples 21 and 22.

[0516] This clearly demonstrates that fusion of UL12 or T7exo allows for 5' to 3' cleavage prior to NHEJ-mediated DNA damage rehabilitation.

[0517] Example 21: Amplicon sequencing was performed using the Cas9-exonuclease fusion protein.

[0518] See Figure 25A -D. Using from Example 20 / Figure 24 Primer pair P1 with a 5' adaptor was used to perform mid-target amplification using the same genomic DNA (donor-free Cas9 and Cas9-exo fusion protein) from Example 20 as a template. The adaptor acts as the binding anchor for the index primer in amplicon sequencing. Cas9-mediated cleavage primarily resulted in precise deletions between cleavage sites. Cas9-mediated PAM-out directional cleavage also resulted in a significant number of small 2nt deletions (1nt shift per cleavage site). Cas9-exonuclease fusions generally resulted in larger deletions. The maximum deletion size was similar among different fusion exonucleases, while UL12- and T7-fused Cas9 showed a higher frequency. See also Figure 25A -D.

[0519] Example 22: Amplicon sequencing was performed using the Cas12a-exonuclease fusion protein.

[0520] Using primer pair P1 with a 5' adaptor, genomic DNA from Example 20 (donor-free Cas12a and Cas12a-exonuclease fusion protein) was used as a template for mid-target amplification. The adaptor acts as the binding anchor for the index primers of amplicon sequencing. Typically, Cas12a results in fragment deletions between dual crRNA cleavage sites and several smaller mid-target deletions. Compared to Cas12a WT, the fusion of the exonuclease with Cas12a results in an increased number of insertions and deletions with larger deletions. See also Figure 26A -D.

[0521] Example 23: Sequence alignment of the alkaline exonucleases homologous to UL12 tested.

[0522] In Figure 15The amino acid sequences of exonucleases identified as homologous to UL12 were compared. This comparison was conducted in... Figures 27A to 27C The results are given in Example 12 and... Figure 11-12 The analysis revealed exonuclease-specific motifs / sequence segments exhibiting high HDR efficiency. Exonucleases PiE, PapE, UL12-1, and UL12-2 demonstrated the highest HDR efficiency in Example 12 and were selected for analysis. This analysis identified PiE (SEQ ID NO: 44), PapE (SEQ ID NO: 43), UL12-1 (SEQ ID NO: 32), and UL12-2 (SEQ ID NO: 33) as sharing a common sequence motif / sequence segment with the amino acid sequence SEQ ID NO: 56 (PXPLMXFXEAATQXQXXXQLWXLLRRGLXTAXTLXWGXXGPXFXXXWLXXXXXXXXXXXXXAXXFGRXNEXXARXXLFRYCVGRAD), wherein the X at position 2 is preferably R or K, the X at position 8 is preferably F or Y, the X at position 17 is preferably D or E, and the X at position 35 is preferably R or K. In SEQ ID NO: 56 and all other amino acid sequence segments contained in this application, X represents any one of the 20 amino acids in the standard genetic code, where an amino acid residue must be present at position X, as X cannot be without an amino acid residue. Therefore, X in SEQ ID NO: 56 represents a variable amino acid residue, while non-X residues in SEQ ID NO: 56 are fixed to the residues indicated by the single-letter codon.

[0523] from Figure 27A and 27B It is evident that UL12-group specific motifs I and II can be identified. These motifs are present in PiE, PapE, UL12-1, and UL12-2, but not in BGLF5, SOX, MD5, DUMAS, and AB4P. In particular, UL12-group specific motif II, composed of SEQ ID NO: 54 (FRYCVGRAD), distinguishes PiE, PapE, UL12-1, and UL12-2 from BGLF5, SOX, MD5, DUMAS, and AB4P. Since UL12-group specific motifs I and II are also included in SEQ ID NO: 56, SEQ ID NO: 56 will also be able to distinguish PiE, PapE, UL12-1, and UL12-2 from BGLF5, SOX, MD5, DUMAS, and AB4P. This means that SEQ ID NO: 56 is present in Example 12 / Figure 11-12 Among all exonucleases exhibiting high HDR efficiency, and not present in Example 12 / Figure 11-12Among all exonucleases that exhibit low HDR efficiency, SEQ ID NO: 56 explains why PiE, PapE, UL12-1, and UL12-2 show higher HDR efficiency compared to BGLF5, SOX, MD5, DUMAS, and AB4P.

[0524] Example 24: PapE-specific amino acid sequence segments

[0525] In Example 12 / Figure 11-12 In this study, PapE (SEQ ID NO: 43) exhibited particularly high HDR efficiency. The amino acid sequences homologous to PapE were compared with those of PapE (SEQ ID NO: 43) to identify sequence motifs / segments specific to the PapE-group exonucleases. Figure 28A and 28B As shown, motifs specific to PapE and PapE-group exonucleases can be identified (PapE-group specific motifs I to IV). PapE-group specific motifs I to IV connect PapE or PapE-group exonucleases with... Figures 27A to 27C Distinguish between UL12-1, UL12-2, PiE, BGLF5, SOX, MD5, DUMAS, and AB4P as shown in the diagram. Figure 28A and 28B The PapE-group specific motifs I, II, III, and IV are contained in at least one of SEQ ID NO:47 (APAESVHACGVL), SEQ ID NO:48 (APAASVHACGVL), SEQ ID NO:49 (AKYAFDPADAGXXVVAAHRRL), SEQ ID NO:50 (APASAPAAVRAA), and SEQ ID NO:51 (LIITPVRXDAA). Therefore, any one of SEQ ID NO:47, 48, 49, 50, and 51 is an amino acid segment specific to PapE (SEQ ID NO:43) or a PapE-group specific exonuclease.

[0526] Example 25: Sequence alignment of the tested T7 exonuclease homologs

[0527] like Figure 29 As shown, the amino acid sequences of exonucleases homologous to T7 were compared. Amino acid sequence motifs / segments (T7 exonuclease motifs I and II) present in SpiPhage, T7, and ME15 but absent in RalTL1, PaPHBO2, or YerO3-12 were identified. Since SpiPhage, T7, and ME15 are present in Example 13 / Figure 13-14The T7 exonuclease-specific motifs I and II showed higher HDR efficiency than RalTL1, PaPHBO2, or YerO3-12, thus the higher HDR efficiency of SpiPhage, T7, and ME15 was correlated with these motifs. Specifically, ME15 (SEQ ID NO: 45) exhibited the highest HDR efficiency among the tested T7 homologs. Figure 14 ME15-specific motifs I and II were identified, present in the amino acid sequence of ME15 (SEQ ID NO: 45) but not in the amino acid sequence of T7 (SEQ ID NO: 30). ME15-specific motifs I and II are contained in at least one of SEQ ID NO: 52 (APTESETLWDCI) and SEQ ID NO: 53 (ILRFNDYNIDT). Therefore, the sequence segments of SEQ ID NO: 52 and / or 53 are associated with an increased HDR frequency of ME15 compared to T7 and allow for differentiation between ME15 and T7 exonucleases. On the other hand, the amino acid sequence of SEQ ID NO: 98 (WEEEIWHRCCDHAKAR) is a sequence motif specific to T7, SpiPhage, and ME15.

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[0535] Sprunck S, Rademacher S, Vogler F, Gheyselinck J, Grossniklaus U, Dresselhaus T. Egg cell-secreted EC1 triggers sperm cell activation during double fertilization. Science. 2012; 338:1093–7.

[0536] Overview of protein and amino acid sequences

[0537] SEQ ID NO:1: Polynucleotide sequence of the donor fragment (PGK-GUS): The sequence given above

[0538] SEQ ID NO:2: Polynucleotide sequence of module 2x35Ss-Ω

[0539] SEQ ID NO:3: Multinucleotide sequence of the module AGGT_NLS-SpCas9i-NLS*_GCTT for N-terminal fusion complex

[0540] SEQ ID NO:4: Polynucleotide encoding the 2xLF2 linker

[0541] SEQ ID NO:5: Polynucleotide encoding the 4xLF2 linker

[0542] SEQ ID NO:6: Polynucleotide encoding the linker XTEN144

[0543] SEQ ID NO:7: Polynucleotide encoding the XTEN40 linker

[0544] SEQ ID NO:8: Polynucleotide encoding the linker XTEN16

[0545] SEQ ID NO:9: Polynucleotide encoding T7 exonuclease for N-terminal exonuclease-Cas9 fusion

[0546] SEQ ID NO:10: Polynucleotide encoding T5 exonuclease for N-terminal exonuclease-Cas9 fusion

[0547] SEQ ID NO:11: Polynucleotide encoding UL12 exonuclease for N-terminal exonuclease-Cas9 fusion

[0548] SEQ ID NO:12: Polynucleotide encoding UL12-2 exonuclease for N-terminal exonuclease-Cas9 fusion

[0549] SEQ ID NO:13: Polynucleotide encoding BGLF5 exonuclease for N-terminal exonuclease-Cas9 fusion

[0550] SEQ ID NO:14: Polynucleotide encoding DUMAS exonuclease for N-terminal exonuclease-Cas9 fusion

[0551] SEQ ID NO:15: Polynucleotide encoding MD5 exonuclease for N-terminal exonuclease-Cas9 fusion

[0552] SEQ ID NO:16: Polynucleotide sequence of module AATG_NLS-SpCas9i-NLS_TTCG for C-terminal fusion complex

[0553] SEQ ID NO:17: Polynucleotide sequence of module tOCS

[0554] SEQ ID NO:18: Polynucleotide encoding the 2xLF2 linker

[0555] SEQ ID NO:19: Polynucleotide encoding the 4xLF2 linker

[0556] SEQ ID NO:20: Polynucleotide encoding the XTEN144 linker

[0557] SEQ ID NO:21: Polynucleotide encoding the XTEN40 linker

[0558] SEQ ID NO:22: Polynucleotide encoding the linker XTEN16

[0559] SEQ ID NO:23: Polynucleotide encoding T7 exonuclease for C-terminal exonuclease-Cas9 fusion

[0560] SEQ ID NO:24: Polynucleotide encoding T5 exonuclease for C-terminal exonuclease-Cas9 fusion

[0561] SEQ ID NO:25: Polynucleotide encoding UL12 exonuclease for C-terminal exonuclease-Cas9 fusion

[0562] SEQ ID NO:26: Polynucleotide encoding UL12-2 exonuclease for C-terminal exonuclease-Cas9 fusion

[0563] SEQ ID NO:27:sgRNA transcription unit

[0564] SEQ ID NO:28:sgR-PGK1 spacer

[0565] SEQ ID NO:29:sgR-PGK2 spacer

[0566] SEQ ID NO:30: Amino acid sequence of T7 exonuclease

[0567] MALLDLKQFYELREGCDDKGILVMDGDWLVFQAMSAAEFDASWEEEIWHRCCDHAKARQILEDSIKSYETRKKAWAGAPIVLAFTDSVNWRKELVDPNYKANRKAVKKPVGYFEFLDALFEREEFYCIREPMLEGDDVMGVIASNPSAFG ARKAVIISCDKDFKTIPNCDFLWCTTGNILTQTEESADWWHLFQTIKGDITDGYSGIAGWGDTAEDFLNNPFITEPKTSVLKSGKNKGQEVTKWVKRDPEPHETLWDCIKSIGAKAGMTEEDIIKQGQMARILRFNEYNFIDKEIYLWRP

[0568] SEQ ID NO:31: Amino acid sequence of T5 exonuclease

[0569] MSKSWGKFIEEEEAEMASRRNLMIVDGTNLGFRFKHNNSKKPFASSYVSTIQSLAKSYSARTTIVLGDKGKSVFRLEHLPEYKGNRDEKYAQRTEEEKALDEQFFEYLKDAFELCKTTFPTFTIRGVEADD MAAYIVKLIGHLYDHVWLISTDGDWDTLLTDKVSRFSFTTRREYHLRDMYEHHNVDDVEQFISLKAIMGDLGDNIRGVEGIGAKRGYNIIREFGNVLDIIDQLPLPGKQKYIQNLNASEELLFRNLILVDL

[0570] PTYCVDAIAAVGQDVLDKFTKDILEIAEQ

[0571] Amino acid sequence of SEQ ID NO:32:UL12-1 exonuclease

[0572] MESTVGPACPPGRTVTKRPWALAEDTPRGPDSPPKRPRPNSLPLTTTFRPLPP

[0573] PPQTTSAVDPSSHSPVNPPRDQHATDTADEKPRAASPALSDASGPPTPDIPLSP

[0574] GGTHARDPDADPDSPDLDSMWSASVIPNALPSHILAETFERHLRGLLRGVRA

[0575] PLAIGPLWARLDYLCSLAVVLEEAGMVDRGLGRHLWRLTRRGPPAAADAV

[0576] APRPLMGFYEAATQNQADCQLWALLRRGLTTASTLRWGPQGPCFSPQWLK

[0577] HNASLRPDVQSSAVMFGRVNEPTARSLLFRYCVGRADDGGEAGADTRRFIF

[0578] HEPSDLAEENVHTCGVLMDGHTGMVGASLDILVCPRDIHGYLAPVPKTPLA

[0579] FYEVKCRAKYAFDPMDPSDPTASAYEDLMAHRSPEAFRAFIRSIPKPSVRYF

[0580] APGRVPGPEEALVTQDQAWSEAHASGEKRRCSAADRALVELNSGVVSEVLL

[0581] FGAPDLGRHTISPVSWSSGDLVRREPVFANPRHPNFKQILVQGYVLDSHFPD

[0582] CPPHPHLVTFIGRHRTSAEEGVTFRLEDGAGALGAAGPSKASILPNQAVPIALI

[0583] ITPVRIDPEIYKAIQRSSRLAFDDTLAELWASRSPGPGPAAAETTSSSPTTGRSS

[0584] R

[0585] SEQ ID NO:33: Amino acid sequence of UL12-2 exonuclease

[0586] MAAAATPGAKRPADPARDPDSPPKRPRPNSLDLATVFGPRPAPPHPTSPGAP

[0587] GSHSPQSPPRGQPDGGAPGEKARPASPALSEASSGPPTPDIPLSPGGAHAIDPD

[0588] CSPGPPDPDPMWSASAIPNALPPHILAETFERHLRGLLRGVRSPLAIGPLWAR

[0589] LDYLCSLVVSLEAAGMVDRGLGRHLWRLTRRAPPSAAEAVAPRPLMGFYE

[0590] AATQNQADCQLWALLRRGLTTASTLRWGAQGPCFSSQWLTHNASLRLDAQ

[0591] SSAVMFGRVNEPTARNLLFRYCVGRADAGVNDDADAGRFVFHQPGDLAEE

[0592] NVHACGVLMDGHTGMVGASLDILVCPRDPHGYLAPAPQTPLAFYEVKCRA

[0593] KYAFDPADPGAPAASAYEDLMARRSPEAFRAFIRSIPNPGVRYFAPGRVPGP

[0594] EEALVTQDRDWLDSRAAGEKRRCSAPDRALVELNSGVVSEVLLFGVPDLER

[0595] RTISPVAWSSGELVRREPIFANPRHPNFKQILVQGYVLDSHFPDCPLQPHLVT

[0596] FLGRHRAGAEEGVTFRLEDGRGAPAGRGGAPGPAKASILPDQAVPIALIITPV

[0597] RVEPGIYRDIRRNSRLAFDDTLAKLWASRSPGRGPAAADTTSSSPTAGRSSR

[0598] SEQ ID NO:34: Amino acid sequence of BGLF5 exonuclease

[0599] MADVDELEDPMEEMTSYTFARFLRSPETEAFVRNLDRPPQMPAMRYVYLY

[0600] CLCKQIQEFSGETGFCDFVSSLVQENDSKDGPSLKSIYWGLQEATDEQRTVL

[0601] CSYVESMTRGQSENLMWDILRNGIISSSKLLSTIKNGPTKVFEPAPISTNHYFG

[0602] GPVAFGLRCEDTVKDIVCKLICGDASANRQFGFMISPTDGIFGVSLDLCVNVE

[0603] SQGDFILFTDRSCIYEIKCRFKYLFSKSEFDPIYPSYTALYKRPCKRSFIRFINSI

[0604] ARPTVEYVPDGRLPSEGDYLLTQDEAWNLKDVRKRKLGPGHDLVADSLAA

[0605] NRGVESMLYVMTDPSENAGRIGIKDRVPVNIFINPRHNYFYQVLLQYKIVGD

[0606] YVRHSGGGKPGRDCSPRVNIVTAFFRKRSPLDPATCTLGSDLLLDASVEIPVA

[0607] VLVTPVVLPDSVIRKTLSTAAGSWKAYADNTFDTAPWVPSGLFADDESTP

[0608] SEQ ID NO:35: Amino acid sequence of DUMAS exonuclease

[0609] MARSGLDRIDISPQPAKKIARVGGLQHPFVKTDINTINVEHHFIDTLQKTSPN

[0610] MDCRGMTAGIFIRLSHMYKILTTLESPNDVTYTTPGSTNALFFKTSTQPQEPR

[0611] PEELASKLTQDDIKRILLTIESETRGQGDNAIWTLLRRNLITASTLKWSVSGPV

[0612] IPPQWFYHHNTTDTYGDAAAMAFGKTNEPAARAIVEALFIDPADIRTPDHLT

[0613] PEATTKFFNFDMLNTKSPSLLVGTPRIGTYECGLLIDVRTGLIGASLDVLVCD

[0614] RDPLTGTLNPHPAETDISFFEIKCRAKYLFDPDDKNNPLGRTYTTLINRPTMA

[0615] NLRDFLYTIKNPCVSFFGPSANPSTREALITDHVEWKRLGFKGGRALTELDA

[0616] HHLGLNRTISSRVWVFNDPDIQKGTITTIAWATGDTALQIPVFANPRHANFK

[0617] QIAVQTYVLSGYFPALKLRPFLVTFIGRVRRPHEVGVPLRVDTQAAAIYEYN

[0618] WPTIPPHCAVPVIAVLTPIEVDVPRVTQILKDTGNNAITSALRSLRWDNLHPA

[0619] VEEESVDCANGTTSLLRATEKPLL

[0620] SEQ ID NO:36: Amino acid sequence of MD5 exonuclease

[0621] MELGTKFPLSKSCKDESRKRKRGITIDCDSQILVGDEQSNSTKTKPYDEICENI

[0622] VPNYTFGNYILQKIDPNDCRHSLHPLYHRLFYIADVIKQGISEGSLLENKYSYILETEHILLDESRINNLSPSIHASRWCKMVESLTRLQANSELWHIFRQCLLTASSVKWSPNGTI NTAGLITNDLPSRGQTESILFGTRNESLAKSLIAALCVSQSSVRTIDNSDKKNEFDNTTTGILDIEKYSCGLMIDIRTGMLGASLDMVMCNRNRHGILAPCLTDNNIETYEIKCRFKY AFCPEMRSELSQCYERLMATKTVQWFRRFLYTIDCPCVDYFRPDNYPRAKEALITSDDDWKVGHSAYHAAQSRIKCNEFEMHHLTLNKNMSSRVWLFGEPDLQTNSIYPLLWNTGER VLSIPIFANPRHQNFKQIFLQSYVASGYFGNRKIVPFLATFIGRHRRQTELGRCFSLFVDDTEASEVVYEITPEQAIPVILIITPVIIDNTFYVGIEESGYRAFGELVDHLWAKQCRI

[0623] SEQ ID NO:37: Hairpin nucleotide, based on Nikiforov (2014)

[0624] GGAAGGGCCCGCTGACAGTTTTTCTGTCAGCGGGCCCTTCC

[0625] SEQ ID NO:38: Polynucleotide encoding AATG_NLS-LbCas12a(D156R)-NLS(intron)_TTCG(C-terminal fusion for exonuclease)

[0626] SEQ ID NO:39: Polynucleotide sequence of the Cas12a module AGGT_NLS-Cas12a(D156R)-NLS(intron)_GCTT for N-terminal fusion.

[0627] SEQ ID NO:40: Amino acid sequence of NLS-LbCas12a(D156R)-NLS for the C-terminal fusion complex

[0628]

[0629]

[0630] SEQ ID NO 41: Amino acid sequence of the protein NLS-LbCas12a(D156R)-NLS (intron) for N-terminal fusions

[0631] GLQPKKKRKVGGVDSSAAATMSKLEKFTNCYSLSKTLRFKAIPVGKTQENID

[0632] NKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKK

[0633] TRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEI

[0634] ALVNSFNGFTTAFTGFFRNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEK

[0635] VDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVT

[0636] ESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDE

[0637] EVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFG

[0638] EWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYA

[0639] DADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIM

[0640] KDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRN

[0641] YVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMD

[0642] KKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPS

[0643] EDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETE

[0644] KYKDIAGFYREVEEQGYKVSFESASKEVDKLVEEGKLYMFQIYNKDFSDK

[0645] SHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANS

[0646] PIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVR

[0647] VLLKHDDNPYVIGIDRGERNLLYIVVVDKGGNIVEQYSLNEIINNFNGIRIKT

[0648] DYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKISELVEKYDAVI

[0649] ALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALK

[0650] GYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKK

[0651] FISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPK

[0652] KNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLLEQSDKAFYSSFMAL

[0653] MSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADAN

[0654] GAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAA

[0655] TKKAGQAKKKKGSSEQ ID NO 42: Amino acid sequence of NLS-SpCas9-NLS for the N-terminal and C-terminal fusion complex.

[0656]

[0657] SEQ ID NO:43: The amino acid sequence of PapE, a deoxyribonuclease (AHM96060.1) derived from Papiineα herpesvirus 2.

[0658] MQTTTPVDPPSSRSENKRPPAPAGDEGAGPGRGLVDPARPPKRPRPDSLPLAAVCRPATPPSPGRPETPPTPDLPLSPRGTHGIAAPAGEPEPGSPSLLENYVPPAPDAGDAGSTPEPGWSAVAIPDALPSHVLAETFERHL CGLLRGVRRPLDVEPLRARLGYLFSLATALEEAGMVDRGVGGHLLRLSRRAAAADPRPLMAFFEAATQNQAESQLWGLLRRGLTTTASTLKWGPRGPCFSPRWLKNNGDPRLDFQSSAVMFGRTNEPAARALLFRYCVGRADD RDAEGDDAGRRFVFCEPGDAPAASVHACGVLVDAHTGMVGASLDILVCPRDRHGCLNPAPGTPLRFYEVKCRAKYAFDPADAGDPVVAAHRRLVARRSPSDFRAFLRSIARPGVRYFAPGRVPGPEEALVSDHAVWADARAG DEKRRCSALDRALVGLNSGVASDVLLFGDPDPERRTVSPLAWSSGALVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHPHLVTFIGRHRTPDEEGLSLRLEDAPASAPAAVRAAAGASILPDQAVPVALIITPVRVDA

[0659] AIYDLIRRNSRLAFDETLARLWASRAPASDPAAAGETSS

[0660] SEQ ID NO:44: The amino acid sequence of PiE, a deoxyribonuclease (BBM13184.1) derived from Laili flying fox-associated alpha herpesvirus.

[0661] MTSTSSSSPLHPPSPQKRKSLSADGVAGLATPTKRARPHSLPPLVLHWSPPSP

[0662] SLLPQDGTFIFPSDDTKTRAAEETGPPAPQSQNAPVSPLGDKFSPACAPTSGLI

[0663] CSDSEETEDLVESTQVLSSEAATPLSRVEACDLPPPLMWSATSIPNALPPEIFT

[0664] KTFAKYLRKLLIGIDHPLDIEPLQARLGYLYSLMRALEGGGMLSEGLSRYLIC

[0665] QSRPQASRSKLPRPGLPVVNPKPLMRFFEAATQSQGDSQLWALLRRGLATA

[0666] TTLKWGSQGPAFAPQWLDGVVDQSAGGKGAAIAFGRINELTARTILFRYCV

[0667] GRADHTADADPEERFIFHQPDDMAEENVHTCGVLMDTHTGMVGASLDILV

[0668] CPRDQHGCLSPPPKYPLAFYEVKCRAKYAFDPMDLQSPTTLAYNQLMDRRS

[0669] PAAFRRFMHSISKPGVQFISHGNFPGPEEALVTTSSLWDQSSGAPTKKRRCPA

[0670] AEQALVKLNKSVTSSILLFGTPDLEQRTITPVRWDSGCLFYREALFANPRHPN

[0671] FRQILVQAYVLTSHFPDSPVSPHLVTFIGRQRTVAEEGVNFWLETPSPSVACP

[0672] PNHDPSPPPVSNRASIAADQAIPVAVIITPVRLDVAVYKVLQRNSRLAFDATL

[0673] AQLWASRTPKSVLAADETSSSPTTESP

[0674] SEQ ID NO:45: Amino acid sequence of ME15

[0675] MAVLSLKEFRDIRKGCDDKGILVMDGDWLVFQAMSAAEFDASWEEEIWHR

[0676] CCDHAKARQILDDSIKSYSTRKKAWNGAPIVLAFTDTINWRKELVDPTYKEN

[0677] RKATKKPVGYFEFLDALFERPEFYCVREDMLEGDDVMGIIGSNPSAFGARKA

[0678] VIISCDKDFKTIPDCDFLWCTTGNILTQTQESADWWHLFQTIKGDITDGYSGI

[0679] AGWGDSAEGFLNAPFITEPQVSVLKSGKNKGQEVTKWVKRAPTESETLWDC

[0680] IVSIGAKAGMTEEDVIKQGQMARILRFNDYNIDTKEITLWRPSASF

[0681] SEQ ID NO:46: Amino acid sequence of SpiPh (Spi-Phage)

[0682] MSIKSLAQFEAMGLSGKGLLVMDGDWLVFQAMSAAEFDASWEEEIWHRCC

[0683] DHAKARQILDQSISGYANRKKAWVGAPIVLAFTSDTNWRKDVLESYKSNRK

[0684] KTKKPVGYFEFLDAVFEDDRYICVREDNLEGDDVMGIIGSNPVPFGFKKAVL

[0685] VSCDKDFKTIPNCDFFHVTAGKLLEQNEKSADYWWMFQTIKGDITDGYSGI

[0686] AGMGETGALEFLNAPYKLVQETSLIKAGKNKGQERTVWTKRELEESDSLW

[0687] DAIKSMGAKAGMSEEDVRAQALVARILRHNDYNWIDREIYFPEI

[0688] SEQ ID NO:47:APAESVHACGVL

[0689] SEQ ID NO:48:APAASVHACGVL

[0690] SEQ ID NO:49:AKYAFDPADAGXXVVAAHRRL

[0691] SEQ ID NO:50:APASAPAAVRAA

[0692] SEQ ID NO:51:LIITPVRXDAA

[0693] SEQ ID NO:52:APTESETLWDCI

[0694] SEQ ID NO:53:ILRFNDYNIDT

[0695] SEQ ID NO:54:FRYCVGRAD

[0696] SEQ ID NO:55:PXPLMXFFEAATQ

[0697] SEQ ID NO:56:

[0698] PXPLMXFXEAATQXQXXXQLWXLLRRGLXTAXTLXWGXXGPXFXXXWLX

[0699] XXXXXXXXXXXXAXXFGRXNEXXARXXLFRYCVGRAD

[0700] SEQ ID NO:57:AGGT_NLS-LbCas12a(D156R)i-NLS*_GCTT(CDS; Intron;

[0701]

[0702]

[0703]

[0704]

[0705] SEQ ID NO:58: Polynucleotide encoding AATG_SOX_TTCG exonuclease for N-terminal exonuclease-Cas9 fusion

[0706]

[0707]

[0708] SEQ ID NO:59: Polynucleotide encoding AATG_AB4P_TTCG exonuclease for N-terminal exonuclease-Cas9 fusion

[0709]

[0710]

[0711]

[0712] SEQ ID NO:60: Polynucleotide encoding AATG_PapE_TTCG exonuclease for N-terminal exonuclease-Cas9 fusion

[0713]

[0714]

[0715] SEQ ID NO:61: Polynucleotide encoding AATG_PiE_TTCG exonuclease for N-terminal exonuclease-Cas9 fusion

[0716]

[0717]

[0718] SEQ ID NO:62: Polynucleotide sequence of the LbCas12a(D156R) module AATG_NLS-LbCas12ai-NLS_TTCG for C-terminal fusion.

[0719]

[0720]

[0721]

[0722]

[0723] SEQ ID NO:63: A polynucleotide encoding the AATG_ME15*_GCTT exonuclease for the C-terminal exonuclease-Cas9 fusion (* indicates a stop codon).

[0724]

[0725]

[0726] SEQ ID NO:64: A polynucleotide encoding AATG_O3-12*_GCTT exonuclease for the C-terminal exonuclease-Cas9 fusion (* indicates a stop codon).

[0727]

[0728] SEQ ID NO:65: A polynucleotide encoding the AATG_SpiPh*_GCTT exonuclease for the C-terminal exonuclease-Cas9 fusion (* indicates a stop codon).

[0729]

[0730] SEQ ID NO:66: A polynucleotide encoding the AATG_PhBO2*_GCTT exonuclease for the C-terminal exonuclease-Cas9 fusion (* indicates a stop codon).

[0731]

[0732]

[0733] SEQ ID NO:67: A polynucleotide encoding the AATG_RaTL1*_GCTT exonuclease for the C-terminal exonuclease-Cas9 fusion (* indicates a stop codon).

[0734]

[0735]

[0736] SEQ ID NO:68: Amino acid sequence of SOX exonuclease

[0737] MADVDELEDPMEEMTSYTFARFLRSPETEAFVRNLDRPPQMPAMRYVYLY

[0738] CLCKQIQEFSGETGFCDFVSSLVQENDSKDGPSLKSIYWGLQEATDEQRTVL

[0739] CSYVESMTRGQSENLMWDILRNGIISSSKLLSTIKNGPTKVFEPAPISTNHYFG

[0740] GPVAFGLRCEDTVKDIVCKLICGDASANRQFGFMISPTDGIFGVSLDLCVNVE

[0741] SQGDFILFTDRSCIYEIKCRFKYLFSKSEFDPIYPSYTALYKRPCKRSFIRFINSI

[0742] ARPTVEYVPDGRLPSEGDYLLTQDEAWNLKDVRKRKLGPGHDLVADSLAA

[0743] NRGVESMLYVMTDPSENAGRIGIKDRVPVNIFINPRHNYFYQVLLQYKIVGD

[0744] YVRHSGGGKPGRDCSPRVNIVTAFFRKRSPLDPATCTLGSDLLLDASVEIPVA

[0745] VLVTPVVLPDSVIRKTLSTAAGSWKAYADNTFDTAPWVPSGLFADDESTPSEQ ID NO:69: Amino acid sequence of AB4P exonuclease

[0746] MDSSPVTYSGEPPYKLRRLSPSYPYVSKLRERCASKIETLSEGSARDSLEEED

[0747] VSEAMATGAFLATRLYLPSVLPQRITTLTFLDHFKKSRPLPNSDKRLNPIFYR

[0748] LAYIRDLVGEMELEGIVERGTASRLLGASSPAGFVAGTYTHARDLSKTMSLA

[0749] SVRDAVLAIEAQTRDQSESQLWALLRRGLATASTMKWGALGPQYHPQWCE

[0750] VSTNAKGIPNNPALQFGQTNERTARSLISALYVARSEAATPDLLVDPGCGQC

[0751] FVFDESASVPGDAYACGLLMDARTGVVGASLDMLVCDRDPSGVLSPHSTQT

[0752] TLDFFEIKCRAKYLFDPDLFSPVATAYANLLKHRTAVCLRKFLRSIKNPAVEY

[0753] FAPTSVPGATEALITCNSSWKPREVNETNRRCGDFDRDHIALNLDASSDVWL

[0754] FSEPDLESETITPARWDTGELALSVPVFANPRHPNFKQILVQAYVLSGHFPDH

[0755] QLRPFLVTFIGRHRKRCEEGKTFTICDRPEGSPYNLNEVVHSSCAIPILLFVTPVIVDREGCWEDIEIESLTAFNKTADAIWDSDSPADVSEPTSS

[0756] SEQ ID NO:70: Amino acid sequence of O3-12 (YerO3-12) exonuclease

[0757] MSLITLKDFAEMREGKPMEKGVLVMDGDWLVYQSMAAAEVETDWGDDIWTLECDHAKARSILDSAIESYRTRKKAWSDAMVVLAFTDDVNWRKVLVDETYKENRKATRKPVGYRDFLSKLWERDEFIHIKEDMLEGDDVMGIIGSGHEVFGFKKAVLVSCDKDFKTIPDVDFLWCTTGNILTQTKETADWWHLFQTIKGDMTDGYSGIPGWGDTAEAFLNDPFIVEPVESVLKSGKNKGQTVTKWVKRAPDATETLWDCIKSIGAKAGMTEQEIIKQGQMARILRFEEYNYIDKEIYLWTPRS

[0758] SEQ ID NO:71: Amino acid sequence of PHBO2 (PaPHBO2) exonuclease

[0759] MKFNLNELKDHLKPSKNLLVLDGDWLVFQAMSASEQEVDWGNDIWTLTCDHANALDILQNSIEAWTTRRSTWKNATIVVAFSDDTNWRKDLVDENYKTNRKKTRKPCGYRHFVDTYMEREDTICVVHPNLEADDCMGIIGSGGHHFGTQKVTLISIDKDFRTVPNCDFLWCSTNNILPQDQESADFWHLYQTIKGDITDGYSGIKGWGETAEDFLLDPYMLVRQESTLQSGKNKGQLKVQYVKADKGDNSLWDCIVSLGSKVDMSEEDIIKQARMARILRYSDYDFKNQQVILWTPDKLNQ

[0760] SEQ ID NO:72: Amino acid sequence of RaTL1 / RaITL1 exonuclease

[0761] MSEQRLGLLIDADFLAFQAAANATRVVEWEDGVLTTWANMEDCTQAFLSSLEALTSRNRRWSTAKLIMCFTDDHNWRKDILPSYKANRSGVGKGKPIAYWKLVEWVHQNFECFVRPGLEGDDCMGILSTKPSLVGCTHTVIVSPDKDFKTVPGEFFWMTTGESLVLSEEDANYWHMYQTLMGDTTDGYAGCPGVGPTSAAEFLAEPYIAYEASKVLKSGPRKGEEVTYWTQRPLEAGEDLWDGIVSLFKKAGLTEEDALVQARVARILRASDFDFKAKTPILWERPPKEDVGTD

[0762] SEQ ID NO:73: Figure 6 Sequence shown at the bottom

[0763] CCTC TTTTCTCGAAGCCGCGGTGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCGTACTCCACC TCACCCATCTTTT GATCAACA

[0764] SEQ ID NO:74: Sequence of TMV DNA donor

[0765]

[0766]

[0767]

[0768]

[0769]

[0770] T-DNA left border; RdRP (intron); MP (intron); T-DNA right border

[0771] SEQ ID NO:75:(AHM95988.1:1-617)

[0772] MQTTTPAGPPSSRAEHKRPPAPAGNEGAEPGRGVDESARPPKRPRPDSLPLAAVYRPATPPSPGRPETPPTPDLPLSPRGTHGLAAPAGEPEPESDPEPGSQSLLA

[0773] NYVPPAQDVGGADADAGSTPEPGWSAVAIPDALPPHVLAETFERHLCGLLR

[0774] GVRRPLDVEPLRARLGYLFSLATALEEAGMVDRGIGGHLLRLSRRAAAADP

[0775] RPLMAFFEAATQNQAESQLWGLLRRGLTTASTLKWGPRGPCFSPRWLKNN

[0776] DDPRLDFQSSAVMFGRTNEPAARALLFRYCVGRADDRDAEGDDAGRRFVF

[0777] CEPGDAPAASVHACGVLVDAHTGMVGASLDILVCPRDRHGCLNPAPGTPLR

[0778] FYEVKCRAKYAFDPADAGDPVVAAHRRLVARRSPADFRAFLRSIARPGVRY

[0779] FAPGRVPGPEEALVSDHAVWADARAGDEKRRCSALDRALVGLNSGVESDV

[0780] LLFGDPDPERRTVSPLAWSSGALVHREPIFANPRHPNFKQILVQAYVLASHFP

[0781] ECPLHPHLVTFIGRHRTPDEEGLSLRLEDAPASAPAAVRAAAGASILPDQAVP

[0782] VALIITPVRVDAAIYDLIRRNSRLAFDETLARLWASRAPASDPAAAGGTSS

[0783] SEQ ID NO:76:(AHM96060.1:1-607)

[0784] MQTTTPVDPPSSRSENKRPPAPAGDEGAGPGRGLVDPARPPKRPRPDSLPLA

[0785] AVCRPATPPSPGRPETPPTPDLPLSPRGTHGIAAPAGEPEPGSPSLLENYVPPA

[0786] PDAGDAGSTPEPGWSAVAIPDALPSHVLAETFERHLCGLLRGVRRPLDVEPL

[0787] RARLGYLFSLATALEEAGMVDRGVGGHLLRLSRRAAAADPRPLMAFFEAAT

[0788] QNQAESQLWGLLRRGLTTASTLKWGPRGPCFSPRWLKNNGDPRLDFQSSAV

[0789] MFGRTNEPAARALLFRYCVGRADDRDAEGDDAGRRFVFCEPGDAPAASVH

[0790] ACGVLVDAHTGMVGASLDILVCPRDRHGCLNPAPGTPLRFYEVKCRAKYAF

[0791] DPADAGDPVVAAHRRLVARRSPSDFRAFLRSIARPGVRYFAPGRVPGPEEAL

[0792] VSDHAVWADARAGDEKRRCSALDRALVGLNSGVASDVLLFGDPDPERRTV

[0793] SPLAWSSGALVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHPHLVTFIG

[0794] RHRTPDEEGLSLRLEDAPASAPAAVRAAAGASILPDQAVPVALIITPVRVDA

[0795] AIYDLIRRNSRLAFDETLARLWASRAPASDPAAAGETSS

[0796] SEQ ID NO:77:(AHM96133.1:134-621)

[0797] WSAVAIPDALPPHVLAETFERHLRGLLRGVRRPLDVEPLRARLGYLFSLATA

[0798] LEEAGMVDRGIGGHLLRLSRRAAAADPRPLMAFFEAATQNQAESQLWALL

[0799] RRGLTTASTLKWGPRGPCFSPRWLKNNDDPRLDFQSSAVMFGRTNEPAARA

[0800] LLFRYCVGRADDRDAEGDDAGRRFVFCEPGDAPAASVHACGVLVDAHTGM

[0801] VGASLDILVCPRDRHGCLNPAPGTPLRFYEVKCRAKYAFDPADAGDPVVAA

[0802] HRRLVARRSPADFRAFLRSIARPGVRYFAPGRVPGPEEALVSDHAVWADAR

[0803] AGDEKRRCSALDRALVGLNSGVASDVLLFGDPDPERRTVSPLAWSSGALVH

[0804] REPIFANPRHPNFKQILVQAYVLASHFPECPLRPHLVTFIGRHRTPDEEGLSLR

[0805] LEDAPASAPAAVRAAAGASILPDQAVPVALIITPVRVDAAIYDLIRRNSRLAF

[0806] DETLARLWASRAPASDPAAAGETSS

[0807] SEQ ID NO:78:(AHM96206.1:148-635)

[0808] WSAVAIPDALPPHVLAETFERHLRGLLRGVRRPLDVEPLRARLGYLFSLATA

[0809] LEEAGMVDRGIGGHLLRLSRRAAAADPRPLMAFFEAATQNQAESQLWALL

[0810] RRGLTTASTLKWGPRGPCFSPRWLKNNDDPRLDFQSSAVMFGRTNEPAARA

[0811] LLFRYCVGRADDRDAEGDDAGRRFVFCEPGDAPAASVHACGVLVDAHTGM

[0812] VGASLDILVCPRDRHGCLNPAPGTPLRFYEVKCRAKYAFDPADAGDPVVAA

[0813] HRRLVARRSPADFRAFLRSIARPGVRYFAPGRVPGPEEALVSDHAVWADAR

[0814] AGDEKRRCSALDRALVGLNSGVASDVLLFGDPDPERRTVSPLAWSSGALVH

[0815] REPIFANPRHPNFKQILVQAYVLASHFPECPLRPHLVTFIGRHRTPDEEGLSLR

[0816] LEDAPASAPAAVRAAAGASILPDQAVPVALIITPVRVDAAIYDLIRRNSRLAF

[0817] DETLARLWASRAPASDPAAAGETSS

[0818] SEQ ID NO:79:(AHM96279.1:2-616)

[0819] QTTTPADPPSSRSEHKRPLAPAGDEGAEPGRGPDDPTRPPKRPRPDSLPLAAV

[0820] YRPATPPSPGRPETPPTPDLPLSPRGTHGIAVPAGEPEPESDPEPGSQSLLANY

[0821] VPPAQDVGGADADAGSTPEPGWSAVAIPDALPPHVLAETFERHLRGLLRGV

[0822] RRPLDVEPLRARLGYLFSLATALEEAGMVDRGIGGHLLRLSRRAAAADPRPL

[0823] MAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSPRWLKNNDDP

[0824] RLDFQSSAVMFGRTNEPAARALLFRYCVGRADDRDAEGDDAGRRFVFCEPG

[0825] DAPAASVHACGVLVDAHTGMVGASLDILVCPRDRHGCLNPAPGTPLRFYEV

[0826] KCRAKYAFDPADAGDPVVAAHRRLVARRSPSDFRAFLRSIARPGVRYFAPG

[0827] RVPGPEEALVSDHAVWADARAGDEKRRCSALDRALVGLNSGVASDVLLFG

[0828] DPDPERRTVSPLAWSSGALVHREPIFANPRHPNFKQILVQAYVLASHFPECPL

[0829] HPHLVTFIGRHRTPDEEGLSLRLEDAPASAPAAVRAAGASILPDQAVPVALII

[0830] TPVRVDAAVYDLIRRNSRLAFDETLARLWASRAPASDPAAAGETSS

[0831] SEQ ID NO:80:(AIA09505.1:11-623)

[0832] PPARAESKRPLAPEDEGGDRTRDPGGRSPKRARPNSLPLAAVYRPATPETPE

[0833] RPRTPPTPDLPLSPRGTRALAPPESRAETGPSSPASQSLLANYAPPPSPGDADG

[0834] GDDVDSTVEPGWSAVAIPDALPPHVLAETFESHLRGLLRGVRGPLDVEPLRA

[0835] RLGYLFSLATALEEAGMVDRGLGGHLVRLSRRAPAALGAGAAGGPGAANP

[0836] RPLMAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSPQWLRNN

[0837] HDLRLDCQSAAVMFGRTNEPAARALLFRYCVGRADDEKDEAGRRFVFREP

[0838] GEAPAESVHACGVLMDAHTGMVGASLDILVCPRDQHGCLNPTPGTPLRFYE

[0839] VKCRAKYAFDPADAGEPVVAAHRRLLATRSPVSFRAFVRSIARPGVRYFAP

[0840] GRVPGPEEALVSEHASWADVRAGDEKRRCSAFDRALVGLNSGVASDVLLF

[0841] GDPDLERRTISPLAWDSGELVHREPIFANPRHPNFKQILVQAYVLASHFPECP

[0842] LHPHLVTFIGRHRTLDEEGVSLRLEDAPAAAPAAARAAIRGSILPDQAVPVAL

[0843] IITPVRIDAAVYEVIHRNSRLAFDETLARIWASRDPAPRPAAAGETS

[0844] SEQ ID NO:81:(ARS01649.1:11-594)

[0845] PVARSECKRPLVPDARGGSPKRARPDSLPLVAVHRPASPGTTPERPQTPDLPL

[0846] SPRGTHALAPPGSGPESAPPSPASQSLLANYVPPASPGSTPDAHADVEPGWSA

[0847] VAIPDALPPHVLAETFESHLRGLLRGVRRPLDVEPLRARLGYLFSLVTALEEA

[0848] GMVDRGVGGHLVRLSRRAGAALGAAAANPNPNPRPLMAFFEAATQNQAES

[0849] QLWALLRRGLTTASTLKWGPRGPCFSPQWLRINDDPRLDFQSSAVMFGRTN

[0850] EPAARALLFRYCVGRADDEEDEAGRRFVFREPGDAPAESVHACGVLVDAHT

[0851] GMVGASLDILVCPRDSHGCLNPAPGAPLRFYEVKCRAKYAFDPADAGEAVV

[0852] AAHRRLLATRSPVDFRAFVRSIARPGVRYFAPGRVPGPEEALVSEHAVWAD

[0853] ARAGEEKRRCSAFDRALVGLNSGVASDVLLFGNPDLERRTISPLVWTSGDV

[0854] VHREPIFANPRHANFKQILVQAYVLSSHFPECPLHPHLVTFIGRHRTLDEEGV

[0855] SLRLEDAPGAAPAAVRAAIRGSILPDQAVPVALIITPVRIDAAIYEEIRRNSRL

[0856] AFDETLARIWASR

[0857] SEQ ID NO:82:(ARS01723.1:30-627)

[0858] PKRARPDSLPLVAVYRPASPGTPERPQTPPTPDPPLPPRGASAPAPSGSGSESG

[0859] PPSPASQSLLANYAPPRSPGGDAGSPDAGSPDAGSPDAGSPDAGSPDAGSPD

[0860] AGVEPGWSAAIPDALPPHVLAETFESHLRGLLRGVRRPLDVEPLRARLGYL

[0861] FSLVTALEEAGMVDRGIGGHLIRLSRRAGAAANPRPLMAFFEAATQNQAESQL

[0862] WALLRRGLTTASTLKWGPRGPCFSPQWLRNNDDPRLDFQSAAVMFGRTNE

[0863] PAARALLFRYCVGRADNIGGGDEAGRRFVFREPGDAPAESVHACGVLVDAH

[0864] TGMVGASLDILVCPRDGHGCLNPAGTPLRFYEVKCRAKYAFDPADAGEAV

[0865] VAAHRRLVATRSPVDFRAFVRSIARPGVRYFAPGRVPGPEEALVSDHASWA

[0866] DARAGDEKRRCSAFDRGLVGLNSGVVSDVLLFGEPDLERRTISPLVWTSGG

[0867] VVHREPIFANPRHPNFKQILVQAYVLSSHFPECPLRPHLVTFIGRHRTLDEEG

[0868] VSLRLEDAPAPAPAAARAAIRGSILPDQAVPVALIITPVRIDAAVYDVIRRNSR

[0869] LAFDETLARIWASRDPASRPAAAGETS

[0870] SEQ ID NO:83:(ARS01798.1:30-602)

[0871] PKRARPNSLPLVAVIRPASPGTPERPQTPPPTPDPPLPPRGASAPAPSGSGSGSESG

[0872] PPSPASQSLLANYAPPRSPGGDAGSPDAGVEPGWSAAIPDALPPHVLAETFE

[0873] SHLRGLLRGVRRPLDVEPLRARLGYLFSLVTALEEAGMVDRGIGGHLIRLSR

[0874] RAGAANPRPLMAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSP

[0875] QWLRNNDDPRLDFQSAAVMFGRTNEPAARALLFRYCVGRADDIGGGDAY

[0876] RRFVFREPGDAPAESVHACGVLVDAHTGMVGASLDILVCPRDGHGCLNPAP

[0877] GTPLRFYEVKCRAKYAFDPADAGEAVVAAHRRLVATRSPVDFRAFVRSIAR

[0878] PGVRYFAPGRVPGPEEALVSDHASWADARAGDEKRRCSAFDRGLVGLNSG

[0879] VVSDVLLFGEPDLERRTISPLVWTSGGVVHREPIFANPRHPNFKQILVQAYVL

[0880] SSHFPECPLRPHLVTFIGRHRTLDEEGVSLRLEDAPAPAPAAARAAIRGSILPD

[0881] QAVPVALIITPVRIDAAVYDVIRRNSRLAFDETLARIWASRDPASRPAAAGET

[0882] S

[0883] SEQ ID NO:84:(ARS01873.1:12-624)

[0884] PARAESKRPLVLGDEDENGDRIRDPGHGRSPKRARPNSLPLAAVFRPATPGTP

[0885] KRPRTPPTPDLPLSPRGSRAAASPEFRSETGPSSPASQSLLADYAPPPSPGDAD

[0886] GNDTDSTVEPGWSAVAIPDALPPHVLAETFESHLRGLLRGVRGPLDVEPLRA

[0887] RLGYLFSLATALEEAGMVDRGLGGHLVRLSRRAPAALGAGAAGGPAAANP

[0888] RPLMAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSPQWLRNN

[0889] HDLRLDCQSAAVMFGRTNEPAARALLFRYCVGRADNERDEAGRRFVFREP

[0890] GEAPAESVHACGVLMDAHTGMVGASLDILVCPRDHHGCLNPAPGTPLRFYE

[0891] VKCRAKYAFDPADAGEPVVAAHRRLVATRSPVSFRAFVRSIARPGVRYFAP

[0892] GRVPGPEEALVSEHASWADVRAGDEKRRCSALDRDLVGLNSGVASDVLLF

[0893] GDPDLERRTISPLAWDSGELVHREPIFANPRHPNFKQILVQAYVLASHFPECP

[0894] LHPHLVTFIGRHRTLDEEGVSLRLEDAPAAAPPAARAAIRGSILPDQAVPVAL

[0895] IITPVRIDAAVYEVIRRNSRLAFDETLARIWASRDPVPRPAAADETS

[0896] SEQ ID NO:85:(ARS01948.1:12-624)

[0897] PARAESKRPLVLGDEDENGDRIRDPHGRSPKRARPNSLPLAAVFRPATPGTPE

[0898] RPRTPPTPDLPLSPRGTRAAASPESRSETGPSSPASQSLLADYAPPPSPGDADG

[0899] NDTDSTVEPGWSAVAIPDALPPHVLAETFESHLRGLLRGVRGPLDVEPLRAR

[0900] LGYLFSLATALEEAGMVDRGLGGHLVRLSRRAPAALGAGAAGGPAAANPR

[0901] PLMAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSPQWLRNNH

[0902] DLRLDCQSAAVMFGRTNEPAARALLFRYCVGRADDEKDEAGRRFVFREPGE

[0903] APAESVHACGVLMDAHTGMVGASLDILVCPRDHHGCLNPAPGTPLRFYEVK

[0904] CRAKYAFDPADAGEPVVAAHRRLVATRSPVSFRAFVRSIARPGVRYFAPGR

[0905] VPGPEEALVSEHASWADVRAGDEKRRCSALDRDLVGLNSGVASDVLLFGDP

[0906] DLERRTISPLAWDSGELVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHP

[0907] HLVTFIGRHRTLDEEGVSLRLEDAPAAAPPAARAAIRGSILPDQAVPVALIITP

[0908] VRIDAAVYEVIRRNSRLAFDETLARIWASRDPVPRPAAADETS

[0909] SEQ ID NO:86:(ARS02023.1:12-624)

[0910] PARAESKRPLVLGDEDENGDRIRNPHGRSPKRARPNSLPLAAVFRPATPGTPE

[0911] RPRTPPTPDLPLLPRGTRAAASPESRSETGPSSPASQSLLADYAPPPSPGDADG

[0912] NDTDSTVEPGWSAVAIPDALPPHVLAETFESHLRGLLRGVRGPLDVEPLRAR

[0913] LGYLFSLATALEEAGMVDRGLGGHLVRLSRRAPAALGAGAAGGPAAANPR

[0914] PLMAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSPQWLRNNH

[0915] DLRLDCQSAAVMFGRTNEPAARALLFRYCVGRADDEKDEAGRRFVFREPGE

[0916] APAESVHACGVLMDAHTGMVGASLDILVCPRDHHGCLNPAPGTPLRFYEVK

[0917] CRAKYAFDPADAGEPVVAAHRRLVATRSPVSFRAFVRSIARPGVRYFAPGR

[0918] VPGPEEALVSEHASWADVRAGDEKRRCSALDRDLVGLNSGVASDVLLFGDP

[0919] DLERRTISPLAWDSGELVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHP

[0920] HLVTFIGRHRTLDEEGVSLRLEDAPAAAPPAARAAIRGSILPDQAVPVALIITP

[0921] VRIDAAVYEVIRRNSRLAFDETLARIWASRDPVPRPAAADETS

[0922] SEQ ID NO:87:(ARS02681.1:12-624)

[0923] PARAEIKRPLVLGDEDENGDRIRDPHGRSPKRARPNSLPLAAVFRPATPGTPE

[0924] RPRTPPTPDLPLSPRGTRAAASPESRSETGPSSPASQSLLADYAPPPSPGDADG

[0925] NDTDSTVEPGWSAVAIPDALPPHVLAETFESHLRGLLRGVRRPLDVEPLRAR

[0926] LGYLFSLATALEEAGMVDRGLGGHLVRLSRRAPAALGAGAAGGPAAANPR

[0927] PLMAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSPQWLRNNH

[0928] DLRLDCQSAAVMFGRTNEPAARALLFRYCVGRADDEKDEAGRRFVFREPGE

[0929] APAESVHACGVLMDAHTGMVGASLDILVCPRDHHGCLNPAPGTPLRFYEVK

[0930] CRAKYAFDPADAGEPVVAAHRRLVATRSPVSFRAFVRSIARPGVRYFAPGR

[0931] VPGPEEALVSEHASWADVRAGDEKRRCSALDRDLVGLNSGVASDVLLFGDP

[0932] DLERRTISPLAWDSGELVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHP

[0933] HLVTFIGRHRTLDEEGVSLRLEDAPAAAPPAARAAIRGSILPDQAVPVALIITP

[0934] VRIDAAVYEVIRRNSRLAFDETLARIWASRDPVPRPAAADETS

[0935] SEQ ID NO:88:(ARS02756.1:12-624)

[0936] PARAESKRPLVLGDEDENGDRIRDPHGRSPKRARPNSLPLAAVFRPATPGTPE

[0937] RPRTPPTPDLPLSPRGTRAVASPESRSETGPSSPASQSLLADYAPPPTPGDADD

[0938] NDTDSTVEPGWSAVAIPDALPPHVLAETFESHLRGLLRGVRGPLDVEPLRAR

[0939] LGYLFSLATALEEAGMVDRGLGGHLVRLSRRAPAALGAGAAGGPAAANPR

[0940] PLMAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSPQWLRNNH

[0941] DLRLDCQSAAVMFGRTNEPAARALLFRYCVGRADDEKDEAGRRFVFREPGE

[0942] APAESVHACGVLMDAHTGMVGASLDILVCPRDHHGCLNPAPGTPLRFYEVK

[0943] CRAKYAFDPADAGEPVVAAHRRLVATRSPVSFRAFVRSIARPGVRYFAPGR

[0944] VPGPEEALVSEHASWADVRAGDEKRRCSAFDRDLVGLNSGVASDVLLFGDP

[0945] DLERRTISPLAWDSGELVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHP

[0946] HLVTFIGRHRTLDEEGVSLRLEDAPAAAPPAARAAIRGSILPDQAVPVALIITP

[0947] VRIDAAVYEVIRRNSRLAFDETLARIWASRDPVPRPAAADETS

[0948] SEQ ID NO:89:(ARS02831.1:12-624)

[0949] PARAESKRPLVLGDEDENGDRIRDPHGRSPKRARPNSLPLAAVFRPATPGTPE

[0950] RPRTPPTPDLPLSPRGSRAAASPEFRSETGPSSPASQSLLADYAPPPSPGDADG

[0951] NDTDSTVEPGWSAVAIPDALPPHVLAETFESHLRGLLRGVRGPLDVEPLRAR

[0952] LGYLFSLATALEEAGMVDRGLGGHLVRLSRRAPAALGAGAAGGPAAANPR

[0953] PLMAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSPQWLRNNH

[0954] DLRLDCQSAAVMFGRTNEPAARALLFRYCVGRADNERDEAGRRFVFREPGE

[0955] APAESVHACGVLMDAHTGMVGASLDILVCPRDHHGCLNPAPGTPLRFYEVK

[0956] CRAKYAFDPADAGEPVVAAHRRLVATRSPVSFRAFVRSIARPGVRYFAPGR

[0957] VPGPEEALVSEHASWADVRAGDEKRRCSALDRDLVGLNSGVASDVLLFGDP

[0958] DLERRTISPLAWDSGELVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHP

[0959] HLVTFIGRHRTLDEEGVSLRLEDAPAAAPPAARAAIRGSILPDQAVPVALIITP

[0960] VRIDAAVYEVIRRNSRLAFDETLARIWASRDPVPRPAAADETS

[0961] SEQ ID NO:90:(ARS02906.1:12-624)

[0962] PARAESKRPLVLGDEDENGDRIRDPHGRSPKRARPNSLPLAAVFRPATPGTPE

[0963] RPRTPPTPDLPLSPRGTRAAASPESRSETGPSSPASQSLLADYAPPPSPGDADG

[0964] NDTDSTVEPGWSAVAIPDALPPHVLAETFESHLRGLLRGVRRPLDVEPLRAR

[0965] LGYLFSLATALEEAGMVDRGLGGHLVRLSRRAPAALGAGAAGGPAAANPR

[0966] PLMAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSPQWLRNNH

[0967] DLRLDCQSAAVMFGRTNEPAARALLFRYCVGRADDEKDEAGRRFVFREPGE

[0968] APAESVHACGVLMDAHTGMVGASLDILVCPRDHHGCLNPAPGTPLRFYEVK

[0969] CRAKYAFDPADAGEPVVAAHRRLVATRSPVSFRAFVRSIARPGVRYFAPGR

[0970] VPGPEEALVSEHASWADVRAGDEKRRCSALDRDLVGLNSGVASDVLLFGDP

[0971] DLERRTISPLAWDSGELVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHP

[0972] HLVTFIGRHRTLDEEGVSLRLEDAPAAAPPAARAAIRGSILPDQAVPVALIITP

[0973] VRIDAAVYEVIRRNSRLAFDETLARIWASRDPVPRPAAADETS

[0974] SEQ ID NO:91:(BAC58051.1:12-624)

[0975] PARAESKRPLVLGDEDENGDRIRDPHGRSPKRARPNSLPLAAVFRPATPGTPE

[0976] RPRTPPTPDLPLSPRGSRAAASPEFRSETGPSSPASQSLLADYAPPPSPGDADG

[0977] NDTDSTVEPGWSAVAIPDALPPHVLAETFESHLRGLLRGVRGPLDVEPLRAR

[0978] LGYLFSLATALEEAGMVDRGLGHLVRLSRRAPAALGAGAAGGPAAANPR

[0979] PLMAFFEAATQNQAESQLWALLRRGLTTASTLKKWGPRGPCFSPQWLRNNH

[0980] DLRLDCQSAAVMFGRTNEPAARALLFRYCVGRADNEKDEAGRRFVFREPGE

[0981] APAESVHACGVLMDAHTGMVGASLDILVCPRDHHGCLNPAPGTPLRFYEVK

[0982] CRAKYAFDPADAGEPVVAAHRRLVATRSPVSFRAFVRSIARPGVRYFAPGR

[0983] VPGPEEALVSEHASWADVRAGDEKRRCSALDRDLVGLNSGVASDVLLFGDP

[0984] DLERRTISPLAWDSGELVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHP

[0985] HLVTFIGRHRTLDEEGVSLRLEDAPAAAPPAARAAIRGSILPDQAVPVALIITP

[0986] VRIDAAVYEVIRRNSRLAFDETLARIWASRDPVPRPAAADETS

[0987] SEQ ID NO:92:(NP_851871.1:12-624)

[0988] PARAESKRPLVLGDEDENGDRIRDPGHGRSPKRARPNSLPLAAVFRPATPGTPE

[0989] RPRTPPTPDLPLSPRGTRAAASPESRSETGPSSPASQSLLADYAPPPSPGDADG

[0990] NDTDSTVEPGWSAVAIPDALPPHVLAETFESHLRGLLRGVRGPLDVEPLRAR

[0991] LGYLFSLATALEEAGMVDRGLGGHLVRLSRRAPAALGAGAAGGPAAANPR

[0992] PLMAFFEAATQNQAESQLWALLRRGLTTASTLKWGPRGPCFSPQWLMNNH

[0993] DLRLDCQSAAVMFGRTNEPAARALLFRYCVGRADDEKDEAGRRFVFREPGE

[0994] APAESVHACGVLMDAHTGMVGASLDILVCPRDHHGCLNPAPGTPLRFYEVK

[0995] CRAKYAFDPADAGEPVVAAHRRLVATRSPVSFRAFVRSIARPGVRYFAPGR

[0996] VPGPEEALVSEHASWADVRAGDEKRRCSALDRDLVGLNSGVASDVLLFGDP

[0997] DLERRTISPLAWDSGELVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHP

[0998] HLVTFIGRHRTLDEEGVSLRLEDAPAAAPPAARAAIRGSILPDQAVPVALIITP

[0999] VRIDAAVYEVIRRNSRLAFDETLARIWASRDPVPRPAAADETS

[1000] SEQ ID NO:93:(YP_164454.1:69-615)

[1001] PPTPDLPLSPRGTHGVAAPAGEAEPRLPSPPSPASPSLLADYVPPAPDADADA

[1002] PDVEPWWSAVAIPDALPPHVQAETFERHLRGLLRGVRRPLDVEPLGARLGY

[1003] LFSLATALEEAGMVDRGLGGHLFRLSRRAAAADPRPLMAFFEAATQNQAES

[1004] QLWALLRRGLTTASTLKWGPRGPCFSPRWLKNNDDPRLDFPSSAVMFGRTN

[1005] EPAARALLFRYCVGRTDDRDAEGGEAGRRFVFCEPGDAPVAGVHACGVLV

[1006] DAHTGMVGASLDILVCPRDRHGCLSPTPGTPLRFYEVKCRAKYAFDPADAG

[1007] DPVVAAHRRLVARRSPADFRAFLRSIARPGVRYFAPGQVPGPEEALVSDHAV

[1008] WADARAGGEKRRCPALDRALVGLNSGVASDVLLFGDPDPERRTVSPLVWS

[1009] SGDLVHREPIFANPRHPNFKQILVQAYVLASHFPECPLHPHLVTFIGRHRTPD

[1010] EEGLSLRLGDAPASAPAAVRAAAGASILPDQAVPVALIITPVRVDAAVYDLIR

[1011] RNSRLAFDETLARLWASRAPASDPAVADGTSS

[1012] SEQ ID NO:94:(YP_443858.1:1-617)

[1013] MQTTTPAGPPSSRAEHKRPPAPAGNEGAEPGRGVDESARPPKRPRPDSLPLA

[1014] AVYRPATPPSPGRPETPPTPDLPLSPRGTHGLAAPAGEPEPESDPEPGSQSLLA

[1015] NYVPPAQDVGGADADAGSTPEPGWSAVAIPDALPPHVLAETFERHLCGLLR

[1016] GVRRPLDVEPLRARLGYLFSLATALEEAGMVDRGIGGHLLRLSRRAAAADP

[1017] RPLMAFFEAATQNQAESQLWGLLRRGLTTASTLKWGPRGPCFSPRWLKNN

[1018] DDPRLDFQSSAVMFGRTNEPAARALLFRYCVGRADDRDAEGDDAGRRFVF

[1019] CEPGDAPAASVHACGVLVDAHTGMVGASLDILVCPRDRHGCLNPAPGTPLR

[1020] FYEVKCRAKYAFDPADAGDPVVAAHRRLVARRSPADFRAFLRSIARPGVRY

[1021] FAPGRVPGPEEALVSDHAVWADARAGDEKRRCSALDRALVSLNSGVESDV

[1022] LLFGDPDPERRTVSPLAWSSGALVHREPIFANPRHPNFKQILVQAYVLASHFP

[1023] ECPLHPHLVTFIGRHRTPDEEGLSLRLEDAPASAPAAVRAAAGASILPDQAVP

[1024] VALIITPVRVDAAIYDLIRRNSRLAFDETLARLWASRAPASDPAAAGGTSSSEQ ID NO:95:GVLXDXHTGMVGASLD

[1025] SEQ ID NO:96:EVKCRAKYAFDPXD

[1026] SEQ ID NO:97:FANPRHPNFKQILVQXYVLXXHFP

[1027] SEQ ID NO:98:WEEEIWHRCCDHAKAR

[1028] SEQ ID NO:99:Sequence of TMV transgene

[1029]

[1030]

[1031]

[1032]

[1033]

[1034]

[1035] Right border of T-DNA; Act2p; ω-enhancer RdRP (intron); MP (Intron); GFP ;3`UTR; tNOS ; Left border of T-DNA.

Claims

1. A protein for improving the efficiency of high-performance imaging (HDR) editing of endogenous DNA at target sites in eukaryotic cells or eukaryotic organisms, the protein comprising a site-specific endonuclease and a 5'-3' exonuclease, wherein the site-specific endonuclease is an RNA-guided endonuclease, and wherein the 5'-3' exonuclease is a monomeric 5'-3' exonuclease. The 5'-3' exonuclease of the monomer is a protein with the following amino acid sequence or containing the following amino acid sequence: A) (i) The amino acid sequence defined in SEQ ID NO: 43 (PapE), or B) (i) The amino acid sequence defined in SEQ ID NO: 44 (PiE).

2. A protein for improving the efficiency of high-performance imaging (HDR) of editing endogenous DNA at target sites in eukaryotic cells or eukaryotic organisms, the protein comprising a site-specific endonuclease and a 5'-3' exonuclease, wherein the site-specific endonuclease is an RNA-guided endonuclease, and wherein the 5'-3' exonuclease is a protein with the following amino acid sequence or containing the following amino acid sequence: C) (i) The amino acid sequence defined in SEQ ID NO: 45 (ME15); or D) (i) The amino acid sequence defined in SEQ ID NO: 46 (SpiPh).

3. The protein according to claim 1 or 2, wherein the protein is a fusion protein comprising the site-specific endonuclease and the 5'-3' exonuclease.

4. The protein according to claim 1 or 2, wherein the protein is an oligomeric protein comprising a first protein subunit and a second protein subunit, the first protein subunit comprising the site-specific endonuclease, and the second protein subunit comprising the 5'-3' exonuclease.

5. The protein of claim 4, wherein the first protein subunit comprises the site-specific endonuclease and a first interacting domain, the second protein subunit comprises the 5'-3' exonuclease and a second interacting domain, wherein the first interacting domain and the second interacting domain bind to each other to form the oligomeric protein.

6. The protein according to claim 1 or 2, wherein the protein is an oligomeric protein comprising a first protein subunit, a second protein subunit, a nucleic acid, and an aptamer, wherein the first protein subunit comprises the endonuclease, the second protein subunit comprises the 5'-3' exonuclease, the nucleic acid has a portion capable of binding the endonuclease, and the aptamer is capable of binding the 5'-3' exonuclease.

7. The protein according to claim 6, wherein the nucleic acid is gRNA.

8. The protein according to any one of claims 1 to 6, wherein the site-specific endonuclease is an RNA-guided endonuclease capable of inducing double-strand breaks in DNA or double-strand breaks within DNA, or an RNA-guided endonuclease with nicking enzyme activity capable of inducing single-strand nicks in double-stranded DNA.

9. The protein according to claim 8, wherein the site-specific endonuclease is Cas9 or Cas12a.

10. The protein of claim 8, wherein the site-specific endonuclease is a cleavage enzyme variant of Cas9.

11. The protein according to any one of claims 1 to 10, wherein the 5'-3' exonuclease: In in vitro exonuclease assays, the catalytic efficiency k cat / K m Or, in terms of the number of transformations, it has the same or higher 5'-3' exonuclease activity compared to the T7 exonuclease shown in SEQ ID NO: 30; or In in vitro exonuclease assays, the catalytic efficiency k cat / K m Or, in terms of the number of transformations, it has at least twice the 5'-3' exonuclease activity of the T5 exonuclease shown in SEQ ID NO:

31.

12. The protein according to any one of claims 1 to 11, wherein, Compared to the individual application of the protein when the site-specific endonuclease and the 5'-3' exonuclease do not fuse or form a protein complex when donor nucleic acid is provided, the protein provides an increased frequency of double-strand break repair via homology-mediated repair pathways and / or a higher frequency of gene targeting or gene substitution events.

13. The protein according to claim 1, wherein the amino acid sequence of the 5'-3' exonuclease comprises the amino acid sequence segment of SEQ ID NO:

56.

14. The protein according to claim 1, wherein the amino acid sequence of the 5'-3' exonuclease comprises the amino acid sequence segments of SEQ ID NO: 54 and SEQ ID NO:

55.

15. The protein according to any one of claims 3 to 14, wherein the 5'-3' exonuclease is fused to the N-terminus or C-terminus of the site-specific endonuclease.

16. The protein according to any one of claims 3, 8, and 11 to 15, wherein the site-specific endonuclease and the 5'-3' exonuclease are fused via a polypeptide linker.

17. The protein of claim 16, wherein the polypeptide linker consists of 5 to 300 amino acid residues.

18. The protein of claim 17, wherein the polypeptide linker consists of 10 to 200 amino acid residues.

19. The protein of claim 17, wherein the polypeptide linker consists of 20 to 120 amino acid residues.

20. A nucleic acid molecule comprising a polynucleotide encoding a protein according to any one of claims 1 to 19.

21. A nucleic acid construct, plasmid, or vector comprising a polynucleotide of the nucleic acid molecule of claim 20.

22. A kit comprising: - A nucleic acid molecule comprising a polynucleotide encoding the first protein subunit as described in claim 4 or 5, and a nucleic acid molecule comprising a polynucleotide encoding the second protein subunit as described in claim 4 or 5; or - A nucleic acid molecule comprising a polynucleotide encoding a site-specific endonuclease of the protein-nucleic acid complex of claim 6, and a nucleic acid molecule comprising a polynucleotide encoding a 5'-3' exonuclease of the protein-nucleic acid complex of claim 6, wherein the protein-nucleic acid complex is an oligomeric protein of claim 6 comprising a first protein subunit, a second protein subunit, a nucleic acid, and an aptamer.

23. Prokaryotic or eukaryotic somatic cells, which contain i) The protein according to any one of claims 1 to 19, ii) The nucleic acid molecule of claim 20, iii) The nucleic acid construct, plasmid, or vector as described in claim 21, or iv) The kit according to claim 22.

24. The cell of claim 23, wherein the somatic cell is a eukaryotic cell further comprising donor nucleic acid for homology-mediated DNA repair.

25. The cell of claim 24, wherein the donor nucleic acid comprises, in the following order: - A first homologous arm, which is homologous to a first region flanking the target site in the cell genome on the first side of the target site. - An optional target donor sequence, which is inserted into the genomic DNA of the cell at the target site, and - A second homologous arm, which is homologous to a second region on the second side of the target site.

26. The cell of claim 25, wherein the donor sequence comprises or consists of up to 15,000 nucleotides in length.

27. The cell of claim 25, wherein the donor sequence comprises or consists of up to 10,000 nucleotides in length.

28. The cell of claim 25, wherein the donor sequence comprises or consists of up to 20,000 nucleotides in length.

29. The cell according to any one of claims 23 to 28, further comprising a guide RNA or a guide construct encoding the guide RNA, the guide RNA being capable of binding the site-specific endonuclease and guiding the site-specific endonuclease to a target site in the genome of the cell.

30. The cell according to any one of claims 23 to 29, wherein the somatic cell is a plant cell.

31. A kit for editing endogenous DNA at a target site in eukaryotic cells or eukaryotic organisms, the kit comprising: (a) The donor nucleic acid as defined in claim 25 or a donor construct comprising said donor nucleic acid, and (b) The protein as defined in any one of claims 1 to 19, or the nucleic acid molecule as defined in claim 20, or the nucleic acid construct, plasmid or vector as described in claim 21, or the kit as described in claim 22.

32. The kit according to claim 31, further comprising: (c) Cells or eukaryotic organisms.

33. The kit according to claim 31 or 32, further comprising: (d) A guide RNA that can bind to the site-specific endonuclease and guide the protein to a target site on the endogenous DNA of the cell or organism; or a nucleic acid molecule encoding the guide RNA.

34. A method for modifying endogenous DNA in a eukaryotic cell or eukaryotic organism at a target site, the method comprising providing the cell or organism with: (a) the donor nucleic acid as defined in claim 25, and (b) The protein as defined in any one of claims 1 to 19, or the nucleic acid molecule as defined in claim 20, or the nucleic acid construct, plasmid, or vector as described in claim 21, or the kit as described in claim 22. The modification of endogenous DNA includes inserting a target donor sequence into the endogenous DNA at the target site via homology-mediated repair, and / or includes deleting a sequence in the endogenous DNA at the target site via homology-mediated repair.

35. The method of claim 34, wherein the method is a method of inserting a target donor sequence into the endogenous DNA of a eukaryotic cell or eukaryotic organism at a target site, wherein the target donor sequence contained in the donor nucleic acid is inserted into the endogenous DNA.

36. The method of claim 34 or 35, further comprising providing to the cell or organism: (c) A guide RNA or a nucleic acid encoding the guide RNA, the guide RNA being capable of binding to the site-specific endonuclease and guiding the protein to the target site in the endogenous DNA of the cell or organism.

37. The method according to any one of claims 34 to 36, wherein the donor sequence has a length of up to 15 kbp.

38. The method according to any one of claims 34 to 36, wherein the donor sequence has a length of 20 kbp.

39. A method for modifying endogenous DNA in a eukaryotic cell or eukaryotic organism at a target site, the method comprising providing the cell or organism with: The protein as defined in any one of claims 1 to 19, or the nucleic acid molecule as defined in claim 20, or the nucleic acid construct, plasmid, or vector as described in claim 21, or the kit as described in claim 22. The modified endogenous DNA includes the random deletion of two or more nucleotide sequences in the endogenous DNA upstream and / or downstream of the target site, wherein no donor nucleic acid or donor sequence is provided.

40. Use of the protein of any one of claims 1 to 19 or the kit of claim 22 for gene editing in eukaryotic cells.

41. Eukaryotic somatic cells, produced by any one of claims 34 to 39.

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